XAF genes and polypeptides: methods and reagents for modulating apoptosis

ABSTRACT

The invention provides novel XAF nucleic acid sequences. Also provided are XAF polypeptides, anti-XAF antibodies, and methods for modulating apoptosis and detecting compounds which modulate apoptosis.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit from provisional application 60/052,402filed Jul. 14, 1997, provisional application 60/054,491 filed Aug. 1,1997, and provisional application 60/056,338 filed Aug. 18, 1997.

BACKGROUND OF THE INVENTION

This invention relates to apoptosis, tumor necrosis factor-α (TNF-α)mediated signalling, cell cycle and tumor growth suppression.

Apoptosis is a morphologically distinct form of programmed cell deaththat is important in the normal development and maintenance ofmulticellular organisms. Dysregulation of apoptosis can take the form ofinappropriate suppression of cell death, as occurs in the development ofcancers, or in a failure to control the extent of cell death, as isbelieved to occur in acquired immunodeficiency and certainneurodegenerative disorders.

Some baculoviruses encode proteins termed "inhibitors of apoptosisproteins" (IAPs) because they inhibit the apoptosis that would otherwiseoccur when insect cells are infected by the virus. These proteins arethought to work in a manner that is independent of other viral proteins.The baculovirus IAP genes include sequences encoding a ring zincfinger-like motif (RZF), which may be involved in DNA binding, and twoN-terminal domains that consist of a 70 amino acid repeat motif termed aBIR domain (Baculovirus IAP Repeat).

We have recently discovered a mammalian family of IAP polypeptides.These polypeptides include the human proteins HIAP-1, HIAP-2, and XIAPand their murine homologs. A related protein, NAIP, has also been found.The mammalian IAP levels have been shown to be increased both in cancercells and cells which survive events known to induce apoptosis (e.g.,ischemia). The IAPs have also been shown to block apoptosis triggered bydiverse stimuli. These results are consistent with a role for themammalian IAPs as inhibitors of apoptosis.

The IAP family is now known to include at least two Drosophila proteins,in addition to the original four mammalian homologues (Hay et al., Cell83: 1253-1262, 1995). Although we and others have established that theIAPs can suppress apoptosis in tissue culture model systems theirmechanism of action is still under investigation.

SUMMARY OF THE INVENTION

We have discovered a novel family of genes, the XAFs. Members of the XAFgene family encode proteins that interact with IAPs and are associatedwith apoptosis. Our discovery allows the development of diagnostic,prognostic, and therapeutic compounds and methods for the detection andtreatment of diseases involving apoptosis.

In a first aspect, the invention features substantially pure nucleicacid encoding a XAF polypeptide.

In a second aspect, the invention features substantially pure nucleicacid corresponding to at least ten nucleotides of a nucleic acidencoding a XAF polypeptide, where the nucleic acid is antisense nucleicacid and the antisense nucleic acid is sufficient to decrease XAFbiological activity. In various embodiments of this aspect, theantisense nucleic acid corresponds to at least fifteen nucleotides of anucleic acid encoding a XAF polypeptide, at least thirty nucleotides ofa nucleic acid encoding a XAF polypeptide, or at least 100 nucleotidesof a nucleic acid encoding a XAF polypeptide. In other embodiments, theXAF biological activity is decreased by at least 20%, 40%, 60%, or 80%.In yet another embodiment of this aspect of the invention, the antisensenucleic acid is in a vector where the vector is capable of directingexpression of the antisense nucleic acid in a vector-containing cell.

In a third aspect, the invention features a vector that includes asubstantially pure nucleic acid encoding a XAF polypeptide, where thevector is capable of directing expression of the polypeptide in avector-containing cell.

In another related aspect, the invention features a cell that contains asubstantially pure nucleic acid encoding a XAF polypeptide. In apreferred embodiment of this aspect, the nucleic acid is expressed inthe cell. In various preferred embodiments, the cell is present in apatient having a disease that is caused by excessive or insufficientcell death and the cell is selected from the group that includes afibroblast, a neuron, a glial cell, an insect cell, an embryonic stemcell, a myocardial cell, and a lymphocyte.

In a fifth aspect, the invention features a transgenic animal generatedfrom a cell genetically engineered to lack nucleic acid encoding a XAFpolypeptide, where the transgenic animal lacks expression of the XAFpolypeptide.

In a related aspect, the invention features a transgenic animalgenerated from a cell that contains a substantially pure nucleic acidthat replaces DNA encoding a XAF polypeptide, where the nucleic acid isexpressed in the transgenic animal.

In various embodiments of this aspect, the XAF polypeptide is from amammal (e.g., a human or a rodent). In another embodiment, the nucleicacid is genomic DNA or cDNA, and is operably linked to regulatorysequences for expression of the polypeptide where the regulatorysequences include a promoter (e.g., a constitutive promoter, a promoterinducible by one or more external agents, or a cell-type specificpromoter). In other preferred embodiments, the XAF polypeptide isselected from a group that includes XAF-1, XAF-2 N terminus, XAF-2L andXAF-2S. In another embodiment, the XAF-1 has the amino acid sequence ofSEQ ID NO.: 2 or the nucleic acid sequence of SEQ ID NO.: 1, and mayinclude a deletion of the nucleic acids encoding the carboxy terminalamino acids 173 to 317 of XAF-1 (SEQ ID NO.: 8); or a deletion of thenucleic acids encoding the amino terminal amino acids 1 to 172 of XAF-1(SEQ ID NO.: 7). In another embodiment of this aspect of the invention,the XAF-2 N terminus polypeptide has the amino acid sequence of SEQ IDNO.: 4 or the nucleic acid sequence of SEQ ID NO.: 3. In anotherembodiment, the XAF-2L polypeptide has the amino acid sequence of SEQ IDNO.: 10 or the nucleic acid sequence of SEQ ID NO.: 9. In yet anotherembodiment, the XAF-2S polypeptide has the amino acid sequence of SEQ IDNO.: 12 or the nucleic acid sequence of SEQ ID NO.: 11.

In a seventh aspect, the invention features a method of identifying acompound that modulates apoptosis. The method includes: (a) providing acell that has a XAF gene; (b) contacting the cell with a candidatecompound; and (c) monitoring expression of the XAF gene, where analteration in the level of expression of the XAF gene indicates thepresence of a compound which modulates apoptosis. In one preferredembodiment of this aspect, the alteration that is an increase indicatesthe compound is increasing apoptosis, and the alteration that is adecrease indicates the compound is decreasing apoptosis. In variousembodiments of this aspect, the cell is transformed and the cell is notable to induce apoptosis by expression of p53.

In a related aspect, the invention features another method ofidentifying a compound that is able to modulate apoptosis that includes:(a) providing a cell including a reporter gene operably linked to apromoter from a XAF gene; (b) contacting the cell with a candidatecompound; and (c) measuring expression of the reporter gene, where achange in the expression in response to the candidate compoundidentifies a compound that is able to modulate apoptosis. In onepreferred embodiment of this aspect, the alteration that is an increaseindicates the compound is increasing apoptosis, and the alteration thatis a decrease indicates the compound is decreasing apoptosis. In variousembodiments of this aspect, the cell is transformed and the cell is notable to induce apoptosis by expression of p53.

In a ninth aspect, the invention features a method of identifying acompound that is able to inhibit XAF-mediated apoptosis that includes:(a) providing a cell expressing an apoptosis-inducing amount of XAF; (b)contacting the cell with a candidate compound; and (c) measuring thelevel of apoptosis in the cell, where a decrease in the level relativeto a level in a cell not contacted with the candidate compound indicatesa compound that able to inhibit XAF-mediated apoptosis. In variousembodiments of this aspect, the cell is transformed and the cell is notable to induce apoptosis by expression of p53.

In a tenth aspect, the invention features a method of identifying acompound that is able to induce XAF-mediated apoptosis that includes:(a) providing a cell expressing an apoptosis-inducing amount of XAF; (b)contacting the cell with a candidate compound; and (c) measuring levelof apoptosis in the cell, where an increase in the level relative to alevel in a cell not contacted with the candidate compound indicates acompound that able to induce XAF-mediated apoptosis. In variousembodiments of this aspect, the cell is transformed and the cell is notable to induce apoptosis by expression of p53.

In related aspects, the invention features other methods of identifyinga compound that is able to modulate apoptosis

One such method includes: (a) providing a cell expressing a TRAFpolypeptide, a XAF polypeptide, and a reporter gene operably linked toDNA that includes an NF-κB binding site; (b) contacting the cell with acandidate compound; and (c) measuring expression of the reporter gene,where a change in expression in response to the compound indicates thatthe compound is able to modulate apoptosis. In a preferred embodiment ofthis aspect of the invention, the TRAF is selected from a group thatincludes TRAF2, TRAF5, and TRAF6. In various embodiments of this aspect,the cell is transformed and the cell is not able to induce apoptosis byexpression of p53.

A second such method includes: (a) providing a cell expressing a TRAFpolypeptide, a XAF polypeptide, an IAP polypeptide, and a reporter geneoperably linked to DNA that includes an NF-κB binding site; (b)contacting the cell with a candidate compound; and (c) measuringexpression of the reporter gene, where a change in expression inresponse to the compound indicates that the compound is able to modulateapoptosis. In a preferred embodiment of this aspect of the invention,the IAP is XIAP. In another preferred embodiment of this aspect of theinvention, the TRAF is selected from a group that includes TRAF2, TRAF5,and TRAF6. In various embodiments of this aspect, the cell istransformed and the cell is not able to induce apoptosis by expressionof p53.

A third such method includes: (a) providing a cell having: (i) areporter gene operably linked to a DNA-binding-protein recognition site;(ii) a first fusion gene capable of expressing a first fusion protein,where the first fusion protein includes a XAF polypeptide covalentlybonded to a binding moiety capable of specifically binding to theDNA-binding-protein recognition site; (iii) a second fusion gene capableof expressing a second fusion protein, where the second fusion proteinincludes a XAF polypeptide covalently bonded to a gene activatingmoiety; (b) exposing the cell to the compound; and (c) measuringreporter gene expression in the cell, where a change in the reportergene expression indicates that the compound is capable of modulatingapoptosis. In a preferred embodiment of this aspect of the invention,the cell is a yeast cell.

A fourth method for detecting a compound capable of modulating apoptosisincludes: (a) providing a cell having: (i) a reporter gene operablylinked to a DNA-binding-protein recognition site; (ii) a first fusiongene capable of expressing a first fusion protein, where the firstfusion protein includes a XAF polypeptide covalently bonded to a bindingmoiety capable of specifically binding to the DNA-binding-proteinrecognition site; (iii) a second fusion gene capable of expressing asecond fusion protein, where the second fusion protein includes an IAPpolypeptide covalently bonded to a gene activating moiety; (b) exposingthe cell to the compound; and (c) measuring reporter gene expression inthe cell, where a change in the reporter gene expression indicates thatthe compound is capable of modulating apoptosis. In a preferredembodiment of this aspect of the invention, the IAP is XIAP. In anotherpreferred embodiment, the cell is a yeast cell.

A fifth such method includes: (a) providing a cell having: (i) areporter gene operably linked to a DNA-binding-protein recognition site;(ii) a first fusion gene capable of expressing a first fusion protein,where the first fusion protein includes an IAP polypeptide covalentlybonded to a binding moiety capable of specifically binding to theDNA-binding-protein recognition site; (iii) a second fusion gene capableof expressing a second fusion protein, where the second fusion proteinincludes a XAF polypeptide covalently bonded to a gene activatingmoiety; (b) exposing the cell to the compound; and (c) measuringreporter gene expression in the cell, where a change in the reportergene expression indicates that the compound is capable of modulatingapoptosis. In a preferred embodiment of this aspect of the invention,the IAP is XIAP. In another preferred embodiment, the cell is a yeastcell.

A sixth such method includes: (a) providing a first XAF polypeptideimmobilized on a solid-phase substrate; (b) contacting the first XAFpolypeptide with a second XAF polypeptide; (c) contacting the first XAFpolypeptide and the second XAF polypeptide with a compound; and (d)measuring amount of binding of the first XAF polypeptide to the secondXAF polypeptide, where a change in the amount relative to an amount notcontacted with the compound indicates that the compound is capable ofmodulating apoptosis.

A seventh method for detecting a compound capable of modulatingapoptosis includes: (a) contacting a XAF polypeptide immobilized on asolid-phase substrate; (b) providing the XAF polypeptide with an IAPpolypeptide; (c) contacting the XAF polypeptide and the IAP polypeptidewith a compound; and (d) measuring amount of binding of the XAFpolypeptide to the IAP polypeptide, where a change in the amountrelative to an amount not contacted with the compound indicates that thecompound is capable of modulating apoptosis. In a preferred embodimentof this aspect of the invention, the IAP is XIAP.

An eighth such method includes: (a) providing an IAP polypeptideimmobilized on a solid-phase substrate; (b) contacting the IAPpolypeptide with a XAF polypeptide; (c) contacting the IAP polypeptideand the XAF polypeptide with a compound; and (d) measuring amount ofbinding of the IAP polypeptide to the XAF polypeptide, where a change inthe amount relative to an amount not contacted with the compoundindicates that the compound is capable of modulating apoptosis. In apreferred embodiment of this aspect of the invention, the IAP is XIAP.

In various preferred embodiments of the seventh to eighteenth methodaspects of the invention, the XAF is XAF-1; the XAF is the N-terminus ofXAF-2; the XAF is XAF-2L, or the XAF is XAF-2S. In other embodiments,the XAF is from a mammal (e.g., a human or a rodent).

In a nineteenth aspect, the invention features a method of increasingapoptosis in a cell by administering to the cell an apoptosis inducingamount of XAF polypeptide or fragment thereof.

In related aspects, the invention includes methods of increasingapoptosis by either providing a transgene encoding a XAF polypeptide orfragment thereof to a cell of an animal such that the transgene ispositioned for expression in the cell; or by administering to the cell acompound which increases XAF biological activity in a cell (e.g., byadministering a polypeptide fragment of a XAF polypeptide, a mutant of aXAF polypeptide, or a nucleic acid encoding a XAF polypeptide, a mutantthereof, or a polypeptide fragment thereof).

In preferred embodiment of the nineteenth, twentieth, and twenty-firstaspects of the invention, the XAF is selected from a group that includesXAF-1, XAF-2 N-terminus, XAF-2L, and XAF-2S. In various preferredembodiments, the XAF is from a mammal (e.g., a human or rodent); thecell is in a mammal (e.g., a human or rodent);

the cell is in an mammal diagnosed as having a condition involvinginsufficient apoptosis, (e.g., a cancer such as breast cancer, uterinecervical carcinoma, gastric carcinoma, ovarian epithelial cancer,pediatric medulloblastoma, lung carcinoma, prostate cancer); and thecell is a peripheral blood leukocyte (e.g., a lymphocyte), a muscle cell(e.g., a myocardial cell), an intestinal cell, an ovarian cell, aplacental cell, or a thymus cell (e.g., a thymocyte).

In a twenty-second aspect, the invention features a method of inhibitingapoptosis in a cell, by administering to the cell anapoptosis-inhibiting amount of XAF polypeptide or fragment thereof.

In related aspects, the invention features a method of inhibitingapoptosis in a cell by providing to the cell a transgene encoding a XAFpolypeptide or fragment positioned for expression in the cell; and amethod of inhibiting apoptosis by administering a compound whichdecreases XAF biological activity (e.g., an antibody which specificallybinds to a XAF polypeptide (e.g., a neutralizing antibody), apolypeptide fragment of a XAF polypeptide, a mutant form of a XAFpolypeptide, an antisense nucleic acid complementary to the XAF codingsequence, a negative regulator of the XAF-dependent apoptotic pathway,or a XAF antisense nucleic acid).

In a preferred embodiment of the twenty-second, twenty-third, andtwenty-fourth aspects of the invention, the XAF is selected from a groupthat includes XAF-1, XAF-2 N-terminus, XAF-2L, and XAF-2S. In variouspreferred embodiments, the XAF is from a mammal (e.g., a human orrodent); the cell is in a mammal (e.g., a human or rodent); and themammal bearing the cell is an mammal diagnosed as having a conditioninvolving excessive apoptosis (e.g., AIDS, a neurodegenerative disease,a myelodysplastic syndrome, or an ischemic injury (caused by, e.g., amyocardial infarction, a stroke, or a reperfusion injury, atoxin-induced liver disease, physical injury, renal failure, a secondaryexsaunguination or blood flow interruption resulting from any otherprimary diseases)). In other preferred embodiments, the cell is a musclecell (e.g., a myocardial cell), a peripheral blood leukocyte (e.g., alymphocyte, such as a T lymphocyte (preferably, a CD4⁺ T lymphocyte)),an intestinal cell, an ovarian cell, a placental cell, a thymus cell(e.g., a thymocyte), or a breast cell.

In the twenty-fifth and twenty-sixth aspects, the invention featuresmethods of diagnosing a mammal for the presence of disease involvingaltered apoptosis or an increased likelihood of developing a diseaseinvolving altered apoptosis. The methods include isolating a sample ofnucleic acid from the mammal and determining whether the nucleic acidincludes a XAF mutation, where the presence of a mutation is anindication that the animal has an apoptosis disease or an increasedlikelihood of developing a disease involving apoptosis; or measuring XAFgene expression in a sample from an animal to be diagnosed, where analteration in the expression or activity relative to a sample from anunaffected mammal is an indication that the mammal has a diseaseinvolving apoptosis or increased likelihood of developing such adisease. In preferred embodiments, XAF gene expression is measured byassaying the amount of XAF polypeptide or XAF biological activity in thesample (e.g., the XAF polypeptide is measured by immunological methods),or XAF gene expression is measured by assaying the amount of XAF RNA inthe sample.

In one preferred embodiment of the twenty-fifth and twenty-sixth of theinvention, the XAF is selected from a group that includes XAF-1, XAF-2N-terminus, XAF-2L, and XAF-2S. In another preferred embodiment, themammal is a human.

In a twenty-seventh aspect, the invention features a kit for diagnosinga mammal for the presence of a disease involving altered apoptosis or anincreased likelihood of developing a disease involving altered apoptosisthat includes a substantially pure antibody that specifically binds aXAF polypeptide.

Another such kit includes a material for measuring XAF RNA (e.g., aprobe). In a preferred embodiment, the material is a nucleic acid probe.

A third such kit includes both a substantially pure antibody thatspecifically binds a XAF polypeptide, as well as a material formeasuring XAF RNA. In a preferred embodiment, the kit also includes ameans for detecting the binding of the antibody to the XAF polypeptide.In another preferred embodiment, the material is a nucleic acid probe.

In a thirtieth aspect, the invention features a method of obtaining aXAF polypeptide, including: (a) providing a cell with DNA encoding a XAFpolypeptide, the DNA being positioned for expression in the cell; (b)culturing the cell under conditions for expressing the DNA; and (c)isolating the XAF polypeptide.

In preferred embodiments of this aspect of the invention, the XAF isXAF-1, XAF-2 N terminus, XAF-2L, or XAF-2S. In another preferredembodiment, the DNA further includes a promoter inducible by one or moreexternal agents.

In a thirty-first aspect, the invention features a method of isolating aXAF gene or portion thereof having sequence identity to human XAF-1. Themethod includes amplifying by polymerase chain reaction the XAF gene orportion thereof using oligonucleotide primers wherein the primers (a)are each greater than 13 nucleotides in length; (b) each have regions ofcomplementarity to opposite DNA strands in a region of the nucleotidesequence of FIG. 1; and (c) optionally contain sequences capable ofproducing restriction endonuclease cut sites in the amplified product;and isolating the XAF gene or portion thereof.

In a related aspect, the invention features a method of isolating a XAFgene or portion thereof having sequence identity to human XAF-2L orXAF-2S. The method includes amplifying by polymerase chain reaction theXAF gene or portion thereof using oligonucleotide primers wherein theprimers (a) are each greater than 13 nucleotides in length; (b) eachhave regions of complementarity to opposite DNA strands in a region ofthe nucleotide sequence of FIG. 37A; and (c) optionally containsequences capable of producing restriction endonuclease cut sites in theamplified product; and isolating the XAF gene or portion thereof.

In another related aspect, the invention features a method of isolatinga XAF gene or fragment thereof from a cell, including the steps of: (a)providing a sample of cellular DNA; (b) providing a pair ofoligonucleotides having sequence homology to a conserved region of a XAFgene; (c) combining the pair of oligonucleotides with the cellular DNAsample under conditions suitable for polymerase chain reaction-mediatedDNA amplification; and (d) isolating the amplified XAF gene or fragmentthereof. In a preferred embodiment of the above three aspects, thepolymerase chain reaction is reverse-transcription polymerase chainreaction (e.g., RACE).

In yet another related aspect, the invention features a method ofidentifying a XAF gene in a mammalian cell that includes: (a) providinga preparation of mammalian cellular DNA; (b) providing adetectably-labeled DNA sequence having identity to a conserved region ofa second known XAF gene; and (c) contacting the preparation of cellularDNA with the detectably-labeled DNA sequence under hybridizationconditions that provide detection of a gene having 50% or greaternucleotide sequence identity to the detectably-labeled DNA sequence; andidentifying the XAF gene. In one preferred embodiment of this method fordetecting a XAF gene, the DNA sequence includes at least a portion ofXAF-1. In another preferred embodiment, the DNA sequence includes atleast a portion of XAF-2L. In another preferred embodiment, the DNAsequence includes at least a portion of XAF-2S.

In a thirty-fifth aspect, the invention features a method foridentifying a XAF gene that includes the steps of: (a) providing amammalian cell sample; (b) introducing by transformation into the cellsample a candidate XAF gene; (c) expressing the candidate XAF genewithin the cell sample; and (d) determining whether the sample exhibitsan altered level of apoptosis, where an alteration in the level ofapoptosis identifies a XAF gene. Preferably, the alteration is anincrease in apoptosis and the cell is a leukocyte, a fibroblast, aninsect cell, a glial cell, a myocardial cell, an embryonic stem cell, ora neuron.

In other aspects, the invention features a XAF nucleic acid for use inmodulating apoptosis, a XAF polypeptide for use in modulating apoptosis,the use of a XAF polypeptidc for the manufacture of a medicament for themodulation of apoptosis, and the use of a XAF nucleic acid for themanufacture of a medicament for the modulation of apoptosis. Preferably,the XAF is selected from a group that includes XAF-1, XAF-2 N terminus,XAF-2L, and XAF-2S.

In a fortieth aspect, the invention features a substantially pureantibody that specifically binds a XAF polypeptide, or a fragment or amutant thereof. In one preferred embodiment of this aspect, the XAFpolypeptide is selected from a group that includes XAF-1, XAF-2 Nterminus XAF-2S, and XAF-2L. In other preferred embodiments, the XAFpolypeptide is from a mammal (e.g., a human or a rodent), and theantibody is a polyclonal antibody, a monoclonal antibody, or aneutralizing antibody.

By "XAF", "XAF protein", or "XAF polypeptide" is meant a polypeptide, orfragment thereof, which has at least 30%, more preferably at least 35%,and most preferably 40% amino acid identity to either the amino-terminal131 amino acids of the human XAF-1 (SEQ ID NO.: 2) or the amino-terminal135 amino acids of human XAF-2L (SEQ ID NO.: 10) polypeptides. It isunderstood that polypeptide products from splice variants of XAF genesequences are also included in this definition. Preferably, the XAFprotein is encoded by nucleic acid having a sequence which hybridizes toa nucleic acid sequences present in either SEQ ID NO.: 1 or SEQ ID NO.:9 under stringent conditions. Even more preferably the encodedpolypeptide also has XAF biological activity. Preferably, the XAFpolypeptide has at least three zinc finger domains. More preferably, theXAF polypeptide has at least six zinc finger domains, at least five ofwhich occur within 150 amino acids of the N-terminus.

By "zinc finger" is meant a binding domain capable of associating withzinc. A preferable zinc binding domain has the amino acid sequence5'C-X₂₋₅ -C-X₁₁₋₁₈ -C/H-X₂₋₅ -C/H 3' (SEQ ID NOS.: 13-140), wherein "X"may be any amino acid. A more preferable zinc binding domain has theamino acid sequence 5' C-X₁₋₂ -C-X₁₁ -H-X₃₋₅ -C 3' (SEQ ID NOS.: 45, 77,109 and 141-143), wherein "X" may be any amino acid. Even morepreferably, a zinc binding domain has the amino acid sequence 5' C-X₂-H-X-H-X₃ -C 3' (SEQ ID NO: 45), wherein "X" may be any amino acid. Mostpreferably, a zinc binding domain is one found in a XAF polypeptide.

By "XAF biological activity" is meant any one or more of the biologicalactivities described herein for XAF-1, XAF-2L, or XAF-2S, including,without limitation, the ability to bind an IAP (e.g., a XIAP), oranother XAF polypeptide; the ability to cause apoptosis when transfectedinto a cell (particularly in a HeLa cell); the ability to enhance theNF-κB inducing activity of a TRAF; and the ability to specifically binda XAF-1, XAF-2L, or XAF-2S specific antibody.

By "modulating apoptosis" or "altering apoptosis" is meant increasing ordecreasing the number of cells that undergo apoptosis (than wouldotherwise be the case) in a given cell population. Preferably, the cellpopulation is selected from a group including T cells, neuronal cells,fibroblasts, myocardial cells, or any other cell line known to undergoapoptosis in a laboratory setting (e.g., the baculovirus infected insectcells or an in vivo assay). It will be appreciated that the degree ofmodulation provided by a XAF polypeptide or a modulating compound in agiven assay will vary, but that one skilled in the art can determine thestatistically significant change or a therapeutically effective changein the level of apoptosis which identifies a XAF polypeptide or acompound which modulates XAF or is a XAF therapeutic.

By "high stringency conditions" is meant hybridization in 2X SSC at 40°C. with a DNA probe length of at least 40 nucleotides. For otherdefinitions of high stringency conditions, see Ausubel, F. et al., 1994,Current Protocols in Molecular Biology, John Wiley & Sons, New York,6.3.1-6.3.6, hereby incorporated by reference.

By "IAP" is meant an amino acid sequence which has identity tobaculoviris inhibitors of apoptosis. Mammalian IAPs include, withoutlimitation, NAIP, HIAP1, HIAP2, and XIAP. Preferably, such a polypeptidehas an amino acid sequence which is at least 45%, preferably 60%, andmost preferably 85% or even 95% identical to at least one of the aminoacid sequences of a baculovirus IAP.

By "inhibiting apoptosis" is meant any decrease in the number of cellswhich undergo apoptosis relative to an untreated control. Preferably,the decrease is at least 25%, more preferably the decrease is 50%, andmost preferably the decrease is at least one-fold.

By "polypeptide" is meant any chain of more than two amino acids,regardless of post-translational modification such as glycosylation orphosphorylation.

By "pharmaceutically acceptable carrier" means a carrier which isphysiologically acceptable to the treated mammal while retaining thetherapeutic properties of the compound with which it is administered.One exemplary pharmaceutically acceptable carrier is physiologicalsaline. Other physiologically acceptable carriers and their formulationsare known to one skilled in the art and described, for example, inRemington's Pharmaceutical Sciences, (18^(th) edition), ed. A. Gennaro,1990, Mack Publishing Company, Easton, Pa.

By "substantially identical" is meant a polypeptide or nucleic acidexhibiting at least 50%, preferably 85%, more preferably 90%, and mostpreferably 95% homology to a reference amino acid or nucleic acidsequence. For polypeptides, the length of comparison sequences willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids. For nucleic acids, the length of comparison sequences willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides.

Sequence identity is typically measured using sequence analysis softwarewith the default parameters specified therein (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine, valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

By "substantially pure polypeptide" is meant a polypeptide that has beenseparated from the components that naturally accompany it. Typically,the polypeptide is substantially pure when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the polypeptide is aXAF polypeptide that is at least 75%, more preferably at least 90%, andmost preferably at least 99%, by weight, pure. A substantially pure XAFpolypeptide may be obtained, for example, by extraction from a naturalsource (e.g., a fibroblast, neuronal cell, or lymphocyte) by expressionof a recombinant nucleic acid encoding a XAF polypeptide, or bychemically synthesizing the protein. Purity can be measured by anyappropriate method, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants which accompany it in itsnatural state. Thus, a protein which is chemically synthesized orproduced in a cellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptides notonly includes those derived from eukaryotic organisms but also thosesynthesized in E. coli or other prokaryotes. By "substantially pure DNA"is meant DNA that is free of the genes which, in the naturally-occurringgenome of the organism from which the DNA of the invention is derived,flank the gene. The term therefore includes, for example, a recombinantDNA which is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or which exists as a separate molecule (e.g., a cDNA or agenomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

By "TRAF" is meant a member of the TRAF family of proteins. TRAF familymembers each possess an amino terminal RING zinc finger and/oradditional zinc fingers, a leucine zipper, and a unique, conservedcarboxy terminal coiled coil motif, the TRAF-C domain, which defines thefamily. TRAF1 and TRAF2 were first identified as components of theTNF-R2 signaling complex (Rothe et al., Cell 78: 681-692, 1994).Preferred TRAF polypeptides are TRAF2, TRAF5, and TRAF6.

By "transgene" is meant any piece of DNA which is inserted by artificeinto a cell, and becomes part of the genome of the organism whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism.

By "transgenic" is meant any cell which includes a DNA sequence which isinserted by artifice into a cell and becomes part of the genome of theorganism which develops from that cell. As used herein, the transgenicorganisms are generally transgenic mammals (e.g., rodents such as ratsor mice) and the DNA (transgene) is inserted by artifice into thenuclear genome.

By "knockout mutation" is meant an alteration in the nucleic acidsequence that reduces the biological activity of the polypeptidenormally encoded therefrom by at least 80% relative to the unmutatedgene. The mutation may, without limitation, be an insertion, deletion,frameshift mutation, or a missense mutation. Preferably, the mutation isan insertion or deletion, or is a frameshift mutation that creates astop codon.

By "transformation" is meant any method for introducing foreignmolecules into a cell. Lipofection, calcium phosphate precipitation,retroviral delivery, electroporation, and biolistic transformation arejust a few of the teachings which may be used. For example, biolistictransformation is a method for introducing foreign molecules into a cellusing velocity driven microprojectiles such as tungsten or goldparticles. Such velocity-driven methods originate from pressure burstswhich include, but are not limited to, helium-driven, air-driven, andgunpowder-driven techniques. Biolistic transformation may be applied tothe transformation or transfection of a wide variety of cell types andintact tissues including, without limitation, intracellular organelles(e.g., and mitochondria and chloroplasts), bacteria, yeast, fungi,algae, animal tissue, and cultured cells.

By "transformed cell" is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding (as used herein) a XAF polypeptide.

By "positioned for expression" is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of, e.g.,a XAF-1 polypeptide, a recombinant protein or a RNA molecule).

By "reporter gene" is meant any gene which encodes a product whoseexpression is detectable. A reporter gene product may have one of thefollowing attributes, without restriction: fluorescence (e.g., greenfluorescent protein), enzymatic activity (e.g., luciferase orchloramphenicol acetyl transferase), toxicity (e.g., ricin), or anability to be specifically bound by a second molecule (e.g., biotin or adetectably labeled antibody).

By "promoter" is meant a minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for cell type-specific, tissue-specific orinducible by external signals or agents; such elements may be located inthe 5' or 3' or intron sequence regions of the native gene.

By "operably linked" is meant that a gene and one or more regulatorysequences arc connected in such a way as to permit gene expression whenthe appropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequences.

By "conserved region" is meant any stretch of six or more contiguousamino acids exhibiting at least 30%, preferably 50%, and most preferably70% amino acid sequence identity between two or more of the XAF familymembers, (e.g., between human XAF-1 and another human XAF).

By "detectably-labeled" is meant any means for marking and identifyingthe presence of a molecule, e.g., an oligonucleotide probe or primer, agene or fragment thereof, or a cDNA molecule. Methods fordetectably-labeling a molecule are well known in the art and include,without limitation, radioactive labeling (e.g., with an isotope such as³² P or ³⁵ S) and nonradioactive labeling (e.g., chemiluminescentlabeling, e.g., fluorescein labeling).

By "antisense," as used herein in reference to nucleic acids, is meant anucleic acid sequence that is complementary to the coding strand of agene, preferably, a XAF gene.

By "purified antibody" is meant antibody which is at least 60%, byweight, free from proteins and naturally occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably 90%, and most preferably at least 99%, byweight, antibody, e.g., a XAF-1, XAF-2 N-terminus, XAF-2L, or XAF-2Sspecific antibody. A purified antibody may be obtained, for example, byaffinity chromatography using recombinantly-produced protein orconserved motif peptides and standard techniques.

By "specifically binds" is meant an antibody that recognizes and binds aXAF polypeptide but that does not substantially recognize and bind othernon-XAF molecules in a sample, e.g., a biological sample, that naturallyincludes protein. A preferred antibody binds to the XAF-1 peptidesequence of FIG. 1 (SEQ ID NO.: 2). Another preferred antibody binds tothe XAF-2 N-terminus peptide sequence of FIG. 35 (SEQ ID NO.: 4). Yetanother preferred antibody binds to the XAF-2L peptide sequence of FIG.37 (SEQ ID NO.: 10). Still another preferred antibody binds to theXAF-2S peptide sequence of FIG. 38C (SEQ ID NO.: 12). A more preferredantibody binds to two or more of XAF-1 (SEQ ID NO.: 2), XAF-2 N-terminus(SEQ ID NO.: 4), XAF-2L (SEQ ID NO.: 10) and XAF-2S (SEQ ID NO.: 12).

By "neutralizing antibodies" is meant antibodies that interfere with anyof the biological activities of a XAF polypeptide, particularly theability of a XAF to participate in apoptosis. The neutralizing antibodymay reduce the ability of a XAF polypeptide to participate in apoptosisby, preferably 50%, more preferably by 70%, and most preferably by 90%or more. Any standard assay of apoptosis, including those describedherein, may be used to assess potentially neutralizing antibodies.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a listing of the cDNA (above; SEQ ID NO.: 1) and predictedamino acid (below; SEQ ID NO.: 2) sequences of human XAF-1.

FIG. 2 is a schematic diagram of the six predicted Zn finger bindingdomains corresponding to the N-terminal 178 amino acids of XAF-1 (SEQ IDNO.: 6).

FIG. 3 is a Northern blot analysis of XAF-1 mRNA in multiple humantissues and various cell lines.

FIG. 4 is a Northern dot-blot analysis of XAF-1 mRNA in multiple adultand fetal human tissues.

FIG. 5 is a genomic Southern blot analysis of XAF-1.

FIG. 6 is a Western blotting analysis of XAF-1 protein expression levelin various cell lines.

FIG. 7 are schematic diagrams of XAF-1 constructs.

FIG. 8 is a Western blotting analysis of XAF-1 (SEQ ID NO.: 2), XAF-1N(SEQ ID NO.: 7) and XAF-1C (SEQ ID NO.: 8) protein expression levelswhen transiently expressed in 293T cells.

FIG. 9 is a graph of showing the effect of p53 and XAF overexpression onsurvival of HEL cells.

FIG. 10 shows the effect of p53 and XAF overexpression on survival ofHeLa cells.

FIGS. 11A, 11B, and 11C show photographs of HEL cells infected withadeno-LacZ, adeno-p53 and adeno-XAF-1, respectively.

FIGS. 12A, 12B, and 12C show photographs of HeLa cells infected withadeno-LacZ, adeno-p53 and adeno-XAF-1, respectively.

FIGS. 13A, 13B, 13C, and 13D are graphs showing cell cycle profiles ofHEL cells transfected with nothing, adeno-LacZ, adeno-p53, andadeno-XAF-1, respectively.

FIGS. 14A, 14B, 14C, and 14D are graphs showing cell cycle profiles ofHeLa cells transfected with nothing, adeno-LacZ, adeno-p53, andadeno-XAF-1, respectively.

FIGS. 15A and 15B show localization of human XAF-1 by FISH. FIG. 15Ashows metaphase spread hybridized with XAF-1 genomic probe. FIG. 15Bshows metaphase spread after G banding with Giemsa stain. Specificfluorescent signals on 17p13.3 are indicated by arrows.

FIGS. 16A and 16B show subcellular localization of the XAF-1 protein.

FIGS. 17A, 17B, and 17C are photographs of CHO-K1 cells expressing greenfluorescent protein (GFP)-labeled XAF-1 visualized with a fluorescentmicroscope.

FIGS. 18A and 18B are photographs of 3Y1 cells expressing GFP visualizedwith a fluorescent microscope.

FIGS. 19A and 19B are photographs of 3Y1 cells expressing GFP-labeledXAF-1 visualized with a fluorescent microscope.

FIG. 20 is a graph of relative luciferase activity induced by NF-κBactivation by expression of indicated proteins.

FIG. 21 is a graph of relative luciferase activity induced by NF-κBactivation by co-expression of indicated proteins.

FIG. 22 is a graph of relative luciferase activity induced by NF-κBactivation by TRAF6 co-expressed with indicated amounts of XAF-1protein.

FIG. 23 is a graph of relative luciferase activity induced by NF-κBactivation by TRAF6 co-expressed with indicated amounts of XIAP protein.

FIG. 24 is a graph of relative luciferase activity induced by NF-κBactivation by TRAF6 co-expressed with XIAP and XAF-1 proteins.

FIG. 25 is a graph of relative luciferase activity induced by NF-κBactivation by TRAF2 co-expressed with XIAP and XAF-1 proteins.

FIG. 26 is a graph of relative luciferase activity induced by NF-κBactivation by TRAF6 co-expressed with either full-length XAF-1 protein,a fragment representing the N-terminus of XAF-1 protein, or a fragmentrepresenting the C-terminus of XAF-1 protein.

FIG. 27 is a graph of relative luciferase activity induced by NF-κBactivation by either TRAF5 or TRAF6 when co-expressed with either XAF-1antisense DNA or Bcl-2 antisense DNA.

FIG. 28 is a graph of relative luciferase activity induced by NF-κBactivation by interleukin-1β (IL-1β) in the presence of either XAF-1antisense RNA or Bcl-2 antisense RNA expression.

FIG. 29 is a graph of relative luciferase activity induced by NF-κBactivation by interleukin-1β (IL-1β) in the presence of DNA encoding forXAF-1 protein.

FIG. 30 is a graph of relative luciferase activity induced by NF-κBactivation by TRAF2, TRAF5, or TRAF6 co-expressed with A20 protein.

FIG. 31 is a graph of relative luciferase activity induced by NF-κBactivation by increasing amounts of A20 protein co-expressed with TRAF6alone, or in combination with XAF-1.

FIG. 32 is a Western blot analysis of myc-tagged proteins fromaffinity-purifications with GST-control and GST-XAF-1 fusion proteins.

FIG. 33 is an autoradiograph of an in vitro binding assay of in vitrotranslated HIAP-1 and TRAF2 proteins with GST-control and GST-XAF-1fusion proteins.

FIG. 34 is a table listing the interaction results of a yeast two-hybridassay.

FIG. 35 is a listing of the cDNA (above; SEQ ID NO.: 3) and thepredicted amino acid (below; SEQ ID NO.: 4) sequences of the N-terminusof human XAF-2. The seven zinc finger motifs are boxed and labeled inRoman numerals.

FIG. 36 is a listing of the 3' untranslated region (UTR) DNA sequence(SEQ ID NO.: 5) of human XAF-2 which is located about 250 base pairsC-terminally of SEQ ID NO.:3.

FIG. 37A is a listing of the full length 5' nucleotide (above; SEQ IDNO.: 9) and amino acid (below; SEQ ID NO.: 10) sequences of the long(XAF-2L) splice variant of XAF-2. The shorter splice variant of XAF-2(XAF-2S) (SEQ ID NO.:1) is spliced as indicated.

FIG. 37B is an alignment comparing the nucleic acid sequence of XAF-2L(SEQ ID NO.:9) (above) with the entire nucleic acid sequence of XAF-2S(below; SEQ ID NO.: 11).

FIGS. 38A, 38B, and 38C are the amino acid sequence listings of XAF-1(SEQ ID NO.:2), XAF-2L (SEQ ID NO.:10), and XAF-2S (SEQ ID NO.: 12),respectively, with the zinc finger binding domains indicated.

FIG. 39 is an alignment comparing the sequence of the first 396 aminoacids of XAF-2L (SEQ ID NO.: 10) (above) with the entire amino acidsequence of XAF-1 (SEQ ID NO.: 2) (below.).

FIG. 40 is a set of two schematic drawings indicating the alignment ofthe zinc finger binding domains in XAF-1 (above) and XAF-2L (below).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

We have discovered a new family of proteins, the XAFs, which interactwith IAPs and are involved the TNFα signal transduction pathway whichregulates apoptosis.

The TNF receptor superfamily includes at least 13 transmembrane type Iglycoproteins composed of two identical subunits with variable numbersof a characteristic cysteine rich extracellular repeat. Included amongthese members are TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2),CD40, Fas, and CD30. The corresponding ligands for these receptors aretypically type II transmembrane glycoproteins expressed on the surfaceof interacting cells. In some instances, notably lymphotoxin-α (alsoknown as TNFβ) and the majority of tumor necrosis factor-α (TNFα), theligand is secreted from the cell.

The signals generated by ligated members of the TNF receptor superfamilycan be stimulatory or inhibitory depending on the nature and activationstate of the target cell. However, there is considerable overlap in thesignal transduction pathways; for instance, ligation of TNF-R1, TNF-R2,CD30, and CD40 (Kitson et al., Nature 384: 372-275, 1996) all result inNF-κB activation, a transcription factor found latent in the cytoplasmof cells complexed to an inhibitor protein termed I-κB. Receptorligation induces the phosphorylation of I-κB, which renders I-κBsusceptible to ubiquitination and subsequent degradation. I-κBdegradation unveils the nuclear translocation signal in NF-κB and allowsnuclear localization and activation of transcription from NF-κBdependent promoters (reviewed in Grilli et al., Int. Rev. Cytol. 143:1-60, 1993).

Tumor necrosis factor-α (TNFα), mediates its diverse effects throughboth the 55-60 kDa TNF-R1 and 75-80 kDa TNF-R2 receptors. Thecytoplasmic domains of TNF-R1 and TNF-R2 are not conserved, which isreflected in both the protein factors associated with the cytoplasmicdomains and in the consequences of receptor stimulation. TNF-α signalingthrough TNF-R2 can induce either proliferative responses (i.e. thymocyteand mononuclear proliferation; Tartaglia et al., Proc. Natl. Acad. Sci.USA 88: 9292-9296, 1991; Tartaglia, et al., J. Immunol. 151: 4637-4641,1993; Gehr et al., J. Immunol. 149: 911-917,1992), or cytolyticresponses (Heller et al., Cell 70: 47-52, 1992; Grell et al., LymphokineCytokine Res. 12: 143-148, 1993) depending upon the cell type andactivation state.

Immunoprecipitation of TNF-R2 complexes and peptide sequence analysis ofthe associated proteins identified HIAP-1 and HIAP-2 as components ofthe unstimulated TNF-R2 signaling complex. Protein-protein interactionanalysis has established that the BIR domains of HIAP-1 and HIAP-2 canbind interchangeably to the TRAF-N domains of TRAF1 and TRAF2 (Rothe etal., Cell 83: 1243-1252, 1995). To date, very little is known regardingthe distribution and function of the protein components of the TNF-R2complex following receptor ligation. Likewise, the functionalconsequences of HIAP-1 and HIAP-2 in the TNF-R2 receptor complex havenot been determined.

The role of HIAP-2 in the TNF-R1 receptor signaling complex has, incontrast, been more clearly defined.

The intracellular domain of TNF-R1 contains an approximately 80 aminoacid protein-protein interaction motif termed a "death domain", which isalso found in the low affinity nerve growth factor and Fas receptors.The cytoplasmic death domain of TNF-R1 does not appear to associate withcomponents of the signal transduction pathways prior to ligand binding.The primary effects of TNF-R1 aggregation are NF-κB activation andapoptosis. These effects are dependent upon interaction of TNF-R1 withTRADD (TNF-R1, associated death domain protein; Hsu et al., Cell 81:495-504, 1995), through their respective death domains. TRADD functionsas an adapter molecule which can recruit a variety of proteins to thesignaling complex. The formation of alternative signaling complexeslikely determines the ultimate fate of the cell.

In certain circumstances, TRADD is capable of triggering the formationof a protein complex called the DISC (Death Inducing Signaling Complex).DISC formation occurs when FADD is recruited to the TNF-R1/TRADDcomplex, again through interaction of death domains (Chinnaiyan et al.,Cell 81: 505-512 1995; Chinnaiyan et al., J. Biol. Chem. 271: 4961-4965,1996). In addition to a carboxy terminal death domain, FADD possesses anamino terminal "death effector domain" (DED), which triggers apoptosisby recruiting FLICE (caspase-8). FLICE possesses an unusually long aminoterminal pro-domain containing two DED homologous sequences which bindto the FADD DED. Bringing FLICE molecules into close proximity resultsin proteolytic auto-activation. The cleavage event that activates FLICEalso releases the enzyme from the DISC, at which point itproteolytically activates other caspases and ultimately results inapoptosis (Muzio et al., Cell 85: 817-827, 1996, Boldin et al., 85:803-815 1996). Dominant-negative mutants of FADD block apoptosis througheither Fas or TNF-R1, indicating that the FADD component is responsiblefor propagating the cell death signal generated through either receptor(Chinnaiyan et al., J. Biol. Chem. 271: 4961-4965, 1996).

However, TNFα binding to TNF-R1 does not result in apoptosis in allcircumstances. The formation of an alternative signaling complexcontributes to the pliability of the TNFα response. The "survivalcomplex" that corresponds to the DISC consists of TRADD bound to TRAF2(TNF receptor associated factor-2) and HIAP-2 (Hsu et al., Immunity 4:387-389, 1996; Hsu et al., Cell 84: 299-308, 1996). HIAP-2 is complexedto TRAF2 prior to TNF-R1 stimulation (Hsu et al., Cell 84: 299-308,1996). This protein interaction may enhance the affinity of TRAF2 forbinding to TRADD, thereby favoring the formation of TRADD/TRAF2complexes rather than the TRADD/FADD/FLICE DISC. Alternatively, HIAP-2may interact with other components of the apoptotic pathway, such as thecaspases, in ways which suppress the apoptotic signals that wouldotherwise be generated.

We have now demonstrated that XAF family members interact with IAPs andare clearly involved in apoptotic and NF-κB inducing signaling pathwaysin mammalian cells. Overexpression of XAF-1 causes cell death intransformed cells. Interestingly, overexpression in non-transformedcells merely leads to growth (cell cycle) arrest. The distinct functionstransformed and merely proliferating cells is surprising andsignificant. Our Western and Northern blot analyses indicate that XAF-1is expressed in a variety of tissues and cell types. Since apoptosis isan event non-specific to any particular cell or tissue type, thesefindings are in keeping with the involvement of the XAF-1 protein inapoptosis in a variety of contexts.

We have also discovered a second XAF family member, XAF-2L. XAF-2L, likeXAF-1, also has seven zinc finger binding domains. A second shorterXAF-2 splice variant, XAF-2S, has also been discovered.

I. The XAF-1 Gene

A yeast 2-hybrid screen of a human placenta cDNA library with XIAP asthe `bait` protein identified a 37 kDa zinc finger protein termed XAF-1(XIAP Associated Factor 1). XAF-1 displays significant homology tomembers of the TRAF family, particularly TRAF6, but lacks the TRAF-C andTRAF-N domains.

II. Synthesis of XAF Proteins

The characteristics of the cloned XAF gene sequences may be analyzed byintroducing the sequence into various cell types or using in vitroextracellular systems. The function of XAF proteins may then be examinedunder different physiological conditions. For example, theXAF-1-encoding DNA sequence may be manipulated in studies to understandthe expression of the XAF-1 gene and gene product. Alternatively, celllines may be produced which over-express the XAF gene product allowingpurification of XAF for biochemical characterization, large-scaleproduction, antibody production, and patient therapy.

For protein expression, eukaryotic and prokaryotic expression systemsmay be generated in which XAF gene sequences are introduced into aplasmid or other vector which is then used to transform living cells.Constructs in which the XAF cDNAs containing the entire open readingframes inserted in the correct orientation into an expression plasmidmay be used for protein expression. Alternatively, portions of the XAFgene sequences, including wild-type or mutant XAF sequences, may beinserted. Prokaryotic and eukaryotic expression systems allow variousimportant functional domains of the XAF proteins to be recovered asfusion proteins and then used for binding, structural and functionalstudies and also for the generation of appropriate antibodies. SinceXAF-1 protein expression increases apoptosis in immortalized cells, itmay be desirable to express the protein under the control of aninducible promoter.

Typical expression vectors contain promoters that direct the synthesisof large amounts of mRNA corresponding to the inserted XAF nucleic acidin the plasmid bearing cells. They may also include eukaryotic orprokaryotic origin of replication sequences allowing for theirautonomous replication within the host organism, sequences that encodegenetic traits that allow vector-containing cells to be selected for inthe presence of otherwise toxic drugs, and sequences that increase theefficiency with which the synthesized mRNA is translated. Stablelong-term vectors may be maintained as freely replicating entities byusing regulatory elements of, for example, viruses (e.g., the OriPsequences from the Epstein Barr Virus genome). Cell lines may also beproduced which have integrated the vector into the genomic DNA, and inthis manner the gene product is produced on a continuous basis.

Expression of foreign sequences in bacteria such as Escherichia colirequires the insertion of the XAF nucleic acid sequence into a bacterialexpression vector. This plasmid vector contains several elementsrequired for the propagation of the plasmid in bacteria, and expressionof inserted DNA of the plasmid by the plasmid-carrying bacteria.Propagation of only plasmid-bearing bacteria is achieved by introducingin the plasmid selectable marker-encoding sequences that allowplasmid-bearing bacteria to grow in the presence of otherwise toxicdrugs. The plasmid also bears a transcriptional promoter capable ofproducing large amounts of mRNA from the cloned gene. Such promoters mayor may not be inducible promoters which initiate transcription uponinduction. The plasmid also preferably contains a polylinker to simplifyinsertion of the gene in the correct orientation within the vector. In asimple E. coli expression vector utilizing the lac promoter, theexpression vector plasmid contains a fragment of the E. coli chromosomecontaining the lac promoter and the neighboring lacZ gene. In thepresence of the lactose analog IPTG, RNA polymerase normally transcribesthe lacZ gene producing lacZ mRNA which is translated into the encodedprotein, β-galactosidase. The lacZ gene can be cut out of the expressionvector with restriction endonucleases and replaced by a XAF genesequence, or fragment, fusion, or mutant thereof. When this resultingplasmid is transfected into E. coli, addition of IPTG and subsequenttranscription from the lac promoter produces XAF mRNA, which istranslated into a XAF polypeptide.

Once the appropriate expression vectors containing a XAF gene, orfragment, fusion, or mutant thereof, are constructed they are introducedinto an appropriate host cell by transformation techniques includingcalcium phosphate transfection, DEAE-dextran transfection,electroporation, micro-injection, protoplast fusion andliposome-mediated transfection. The host cell which are transfected withthe vectors of this invention may be selected from the group consistingof E. coli, pseudomonas, Bacillus subtilus, or other bacilli, otherbacteria, yeast, fungi, insect (using, for example, baculoviral vectorsfor expression), mouse or other animal or human tissue cells. Mammaliancells can also be used to express the XAF-1 protein using a vacciniavirus expression system described in Ausubel et al. (Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., 1994).

In vitro expression of XAF proteins, fusions, polypeptide fragments, ormutants encoded by cloned DNA is also possible using the T7late-promoter expression system. This system depends on the regulatedexpression of T7 RNA polymerase which is an enzyme encoded in the DNA ofbacteriophage T7. The T7 RNA polymerase transcribes DNA beginning withina specific 23-bp promoter sequence called the T7 late promoter. Copiesof the T7 late promoter are located at several sites on the T7 genome,but none is present in E. coli chromosomal DNA. As a result, in T7infected cells, T7 RNA polymerase catalyzes transcription of viral genesbut not of E. coli genes. In this expression system recombinant E. colicells are first engineered to carry the gene encoding T7 RNA polymerasenext to the lac promoter. In the presence of IPTG, these cellstranscribe the T7 polymerase gene at a high rate and synthesize abundantamounts of T7 RNA polymerase. These cells are then transformed withplasmid vectors that carry a copy of the T7 late promoter protein. WhenIPTG is added to the culture medium containing these transformed E. colicells, large amounts of T7 RNA polymerase are produced. The polymerasethen binds to the T7 late promoter on the plasmid expression vectors,catalyzing transcription of the inserted cDNA at a high rate. Since eachE. coli cell contains many copies of the expression vector, largeamounts of mRNA corresponding to the cloned cDNA can be produced in thissystem and the resulting protein can be radioactively labeled. Plasmidvectors containing late promoters and the corresponding RNA polymerasesfrom related bacteriophages such as T3, T5, and SP6 may also be used forin vitro production of proteins from cloned DNA. E. coli can also beused for expression by infection with M 13 Phage mGPI-2. E. coli vectorscan also be used with phage lambda regulatory sequences, by fusionprotein vectors, by maltose-binding protein fusions, and byglutathione-S-transferase fusion proteins.

Eukaryotic expression systems permit appropriate post-translationalmodifications to expressed proteins. Transient transfection of aeukaryotic expression plasmid allows the transient production of a XAFpolypeptide by a transfected host cell. XAF proteins may also beproduced by a stably-transfected mammalian cell line. A number ofvectors suitable for stable transfection of mammalian cells areavailable to the public (e.g., see Pouwels et al., Cloning Vectors: ALaboratory Manual, 1985, Supp. 1987), as are methods for constructingsuch cell lines (see e.g., Ausubel et al., supra). In one example, cDNAencoding a XAF-1 protein, fusion, mutant, or polypeptide fragment iscloned into an expression vector that includes the dihydrofolatereductase (DHFR) gene. Integration of the plasmid and, therefore,integration of the XAF-1-encoding gene into the host cell chromosome isselected for by inclusion of 0.01-300 μM methotrexate in the cellculture medium (as described, Ausubel et al., supra). This dominantselection can be accomplished in most cell types. Recombinant proteinexpression can be increased by DHFR-mediated amplification of thetransfected gene. Methods for selecting cell lines bearing geneamplifications are described in Ausubel et al. (supra). These methodsgenerally involve extended culture in medium containing graduallyincreasing levels of methotrexate. The most commonly usedDHFR-containing expression vectors are pCVSEII-DHFR and pAdD26SV(A)(described in Ausubel et al., supra). The host cells described above or,preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR cells, ATCCAccession No. CRL 9096) are among those most preferred for DHFRselection of a stably-transfected cell line or DHFR-mediated geneamplification.

Eukaryotic cell expression of XAF proteins allows for studies of the XAFgenes and gene products including determination of proper expression andpost-translational modifications for biological activity, identifyingregulatory elements located in the 5' region of XAF genes and theirroles in tissue regulation of XAF protein expression. It also permitsthe production of large amounts of normal and mutant proteins forisolation and purification, and the use of cells expressing XAF proteinsas a functional assay system for antibodies generated against theprotein. Eukaryotic cells expressing XAF proteins may also be used totest the effectiveness of pharmacological agents on XAF associatedapoptosis, or as means by which to study XAF proteins as components of asignal transduction system. Expression of XAF proteins, fusions,mutants, and polypeptide fragments in eukaryotic cells also enables thestudy of the function of the normal complete protein, specific portionsof the protein, or of naturally occurring polymorphisms and artificiallyproduced mutated proteins. The XAF DNA sequences can be altered usingprocedures known in the art, such as restriction endonuclease digestion,DNA polymerase fill-in, exonuclease deletion, terminal deoxynucleotidetransferase extension, ligation of synthetic or cloned DNA sequences andsite-directed sequence alteration using specific oligonucleotidestogether with PCR.

Another preferred eukaryotic expression system is the baculovirus systemusing, for example, the vector pBacPAK9, which is available fromClontech (Palo Alto, Calif.). If desired, this system may be used inconjunction with other protein expression techniques, for example, themyc tag approach described by Evan et al. (Mol. Cell Biol. 5:3610-3616,1985).

Once the recombinant protein is expressed, it can be isolated from theexpressing cells by cell lysis followed by protein purificationtechniques, such as affinity chromatography. In this example, ananti-XAF antibody, which may be produced by the methods describedherein, can be attached to a column and used to isolate the recombinantXAF proteins. Lysis and fractionation of XAF protein-harboring cellsprior to affinity chromatography may be performed by standard methods(see e.g., Ausubel et al., supra). Once isolated, the recombinantprotein can, if desired, be purified further by e.g., by highperformance liquid chromatography (HPLC; e.g., see Fisher, LaboratoryTechniques In Biochemistry And Molecular Biology, Work and Burdon, Eds.,Elsevier, 1980).

Polypeptides of the invention, particularly short XAF-1 fragments andlonger fragments of the N-terminus and C-terminus of the XAF-1 protein,can also be produced by chemical synthesis (e.g., by the methodsdescribed in Solid Phase Peptide Synthesis, 2nd ed., 1984, The PierceChemical Co., Rockford, Ill.). These general techniques of polypeptideexpression and purification can also be used to produce and isolateuseful XAF-1 polypeptide fragments or analogs, as described herein.

Those skilled in the art of molecular biology will understand that awide variety of expression systems may be used to produce therecombinant XAF proteins. The precise host cell used is not critical tothe invention. The XAF proteins may be produced in a prokaryotic host(e.g., E. coli) or in a eukaryotic host (e.g., S. cerevisiae, insectcells such as Sf9 cells, or mammalian cells such as COS-1, NIH 3T3, orHeLa cells). These cells are commercially available from, for example,the American Type Culture Collection, 10,801 University Blvd. Manssas,Va. 20110 (see also Ausubel et al., supra). The method of transformationand the choice of expression vehicle (e.g., expression vector) willdepend on the host system selected. Transformation and transfectionmethods are described, e.g., in Ausubel et al. (supra), and expressionvehicles may be chosen from those provided, e.g. in Pouwels et al.,supra.

III. Testing for the Presence of XAF Biological Activity

Identification of XAF-1 and XAF-2 splice variants allow the study of XAFbiological activity in apoptosis-associated cellular events. Forexample, administration of a XAF-1 protein, or polypeptide fragmentthereof, may have an ability to induce apoptosis, as measured byapoptosis assays known in the art and described herein. Anapoptosis-inhibiting amount of a XAF reagent (e.g., a compound thatreduced the biological function of XAF-1, such as a XAF-1 neutralizingantibody or antisense XAF-1 nucleic acid) may be similarly assessed.Such assays may be carried out in a cell which either expressesendogenous XAF-1, or a cell to which is introduced a heterologous amountof a XAF-1 polypeptide. Preferably, the cell is capable of undergoingapoptosis. Apoptosis or inhibition thereof may be assessed in these XAFexpressing cells, whereby such apoptosis inducing or inhibiting activityis evaluated based upon the level of expression of the XAF polypeptide.

Another approach, which utilizes the activation of the nucleartranscription factor, NF-κB (Kunkel et al., Crit. Rev. Immunol. 9:93-117, 1989) in TNF-mediated signal transduction. In this system therole of a XAF in NF-κB activation may be readily elucidated in variousassays known in the art, such as the I-κB degradation assay. Anothermethod of rapidly measuring NF-κB activity is through the use of areporter gene whose expression is directed by a NF-κB binding sitecontaining promoter (Zeichner et al., J. Virol. 65: 2436-2444, 1991).The expression vector is preferably inserted by artifice into a cellcapable of undergoing apoptosis or is responsive to TNF-receptorfamily-mediated signal transduction. By detecting a change in the levelof expression of the reporter gene, an NF-κB-inducing ability of a XAFmay be readily assessed. This method may also be used to detect anNF-κB-inhibiting ability of a XAF wherein NF-κB activation is stimulatedby another component of the TNF-receptor signalling pathway (e.g.,TRAF6).

It will be understood that these analyses may be undertaken with XAF-1or other XAF proteins (e.g., XAF-2L).

IV. Cellular Distribution of XAF-1

We have looked at the distribution of XAF-1 mRNA expression usingradiolabeled antisense XAF-1 DNA and have found that XAF-1 mRNA isexpressed in at least the following adult tissues: heart, brain,placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus,prostate, testis, ovary, appendix, trachea, small intestine, submucosallining of the colon, and peripheral blood leukocytes. XAF-1 mRNA wasfurther found to be expressed in fetal tissue, including fetal brain,fetal heart, fetal kidney, fetal liver, fetal spleen, fetal thymus, andfetal lung.

V. XAF Fragments

Polypeptide fragments which incorporate various portions of XAF proteinsare useful in identifying the domains important for the biologicalactivities of XAF proteins. Methods for generating such fragments arewell known in the art (see, for example, Ausubel et al., supra) usingthe nucleotide sequences provided herein. For example, a XAF proteinfragment may be generated by PCR amplifying the desired fragment usingoligonucleotide primers designed based upon the XAF-1 (SEQ ID NO.: 1)nucleic acid sequences. Preferably the oligonucleotide primers includeunique restriction enzyme site which facilitate insertion of thefragment into the cloning site of a mammalian expression vector. Thisvector may then be introduced into a mammalian cell by artifice by thevarious techniques known in the art and described herein, resulting inthe production of a XAF gene fragment.

In one approach, XAF-1 polypeptide fragments have been useful inevaluating the portions of the protein involved in NF-κB regulation. Inparticular, polypeptide fragments of the amino- and carboxyl-termini ofXAF-1 protein were used to induce or prevent activity induction byvarious other components of the TNF-receptor signalling pathway (e.g.,TRAF6).

In an alternative approach, polypeptide fragments of various portions ofthe XAF-1 protein are useful in modulating XAF-1 mediated apoptosis, asmay be assessed in the various apoptosis assays known in the art anddescribed herein. XAF-1 polypeptide fragments may be used to alter XAF-1mediated apoptosis by inhibiting binding of the full length XAF-1 to,for example, itself to form XAF-1:XAF-1 homodimers, to another XAFprotein (e.g., XAF-2) to form XAF-1:XAF-2 heterodimers, or to XIAP toform XAF-1:XIAP heterodimers. Preferably, such fragments may include theXAF-1:XAF-1 binding domain, the XAF-1:XAF-2 binding domain or theXAF-1:XIAP binding domain.

VI. XAF Antibodies

In order to prepare polyclonal antibodies, XAF proteins, fragments ofXAF proteins, or fusion proteins containing defined portions of XAFproteins can be synthesized in bacteria by expression of correspondingDNA sequences in a suitable cloning vehicle. Fusion proteins arecommonly used as a source of antigen for producing antibodies. Twowidely used expression systems for E. coli are lacZ fusions using thepUR series of vectors and trpE fusions using the pATH vectors. Theproteins can be purified, and then coupled to a carrier protein andmixed with Freund's adjuvant (to help stimulate the antigenic responseby the animal of choice) and injected into rabbits or other laboratoryanimals. Alternatively, protein can be isolated from XAF expressingcultured cells. Following booster injections at biweekly intervals, therabbits or other laboratory animals are then bled and the sera isolated.The sera can be used directly or can be purified prior to use, byvarious methods including affinity chromatography employing reagentssuch as Protein A-Sepharose, Antigen Sepharose, andAnti-mouse-Ig-Sepharose. The sera can then be used to probe proteinextracts from XAF expressing tissues run on a polyacrylamide gel toidentify XAF proteins. Alternatively, synthetic peptides can be madethat correspond to the antigenic portions of the protein and used toinnoculate the animals.

In order to generate peptide or full-length protein for use in making,for example, XAF-1-specific antibodies, a XAF-1 coding sequence can beexpressed as a C-terminal fusion with glutathione S-transferase (GST;Smith et al., Gene 67: 31-40, 1988). The fusion protein can be purifiedon glutathionc-Sepharose beads, eluted with glutathione, and cleavedwith thrombin (at the engineered cleavage site), and purified to thedegree required to successfully immunize rabbits. Primary immunizationscan be carried out with Freund's complete adjuvant and subsequentimmunizations performed with Freund's incomplete adjuvant. Antibodytiters are monitored by Western blot and immunoprecipitation analysesusing the thrombin-cleaved XAF-1 fragment of the GST-XAF-1 fusionprotein. Immune sera are affinity purified using CNBr-Sepharose-coupledXAF-1 protein. Antiserum specificity is determined using a panel ofunrelated GST proteins (including GSTp53, Rb, HPV-16 E6, and E6-AP) andGST-trypsin (which was generated by PCR using known sequences).

It is also understood by those skilled in the art that monoclonal XAFantibodies may be produced by using as antigen XAF protein isolated fromXAF expressing cultured cells or XAF protein isolated from tissues. Thecell extracts, or recombinant protein extracts, containing XAF protein,may for example, be injected with Freund's adjuvant into mice. Afterbeing injected, the mice spleens may be removed and resuspended inphosphate buffered saline (PBS). The spleen cells serve as a source oflymphocytes, some of which are producing antibody of the appropriatespecificity. These are then fused with a permanently growing myelomapartner cells, and the products of the fusion are plated into a numberof tissue culture wells in the presence of a selective agent such ashypoxanthine, aminopterine, and thymidine (HAT). The wells are thenscreened by ELISA to identify those containing cells making antibodycapable of binding a XAF protein or polypeptide fragment or mutantthereof. These are then re-plated and after a period of growth, thesewells are again screened to identify antibody-producing cells. Severalcloning procedures are carried out until over 90% of the wells containsingle clones which are positive for antibody production. From thisprocedure a stable line of clones which produce the antibody isestablished. The monoclonal antibody can then be purified by affinitychromatography using Protein A Sepharose, ion-exchange chromatography,as well as variations and combinations of these techniques. Truncatedversions of monoclonal antibodies may also be produced by recombinantmethods in which plasmids are generated which express the desiredmonoclonal antibody fragment(s) in a suitable host.

As an alternate or adjunct immunogen to GST fusion proteins, peptidescorresponding to relatively unique hydrophilic regions of, for example,XAF-1 may be generated and coupled to keyhole limpet hemocyanin (KLH)through an introduced C-terminal lysine. Antiserum to each of thesepeptides is similarly affinity purified on peptides conjugated to BSA,and specificity is tested by ELISA and Western blotting using peptideconjugates, and by Western blotting and immunoprecipitation using XAF-1expressed as a GST fusion protein.

Alternatively, monoclonal antibodies may be prepared using the XAFproteins described above and standard hybridoma technology (see, e.g.,Kohler et al., Nature 256: 495, 1975; Kohler et al., Eur. J. Immunol.6:511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; Hammerling etal., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York,N.Y., 1981; Ausubel et al., supra). Once produced, monoclonal antibodiesare also tested for specific XAF protein recognition by Western blot orimmunoprecipitation analysis (by the methods described in Ausubel etal., supra).

Monoclonal and polyclonal antibodies that specifically recognize a XAFprotein (or fragments thereof), such as those described hereincontaining a XAF-1 C-terminal domain, are considered useful in theinvention. They may, for example, be used in an reporter gene assay tomonitor the NF-κB inducing effects (via TRAF6) of a XAF protein.Antibodies that inhibit a XAF-1 described herein may be especiallyuseful in preventing apoptosis in cells undergoing undesirable celldeath or growth arrest.

Antibodies of the invention may be produced using XAF amino acidsequences that do not reside within highly conserved regions, and thatappear likely to be antigenic, as analyzed by criteria such as thoseprovided by the Peptide Structure Program (Genetics Computer GroupSequence Analysis Package, Program Manual for the GCG Package, Version7, 1991) using the algorithm of Jameson and Wolf (CABIOS 4:181, 1988).These fragments can be generated by standard techniques, e.g., by thePCR, and cloned into the pGEX expression vector (Ausubel et al., supra).GST fusion proteins are expressed in E. coli and purified using aglutathione agarose affinity matrix as described in Ausubel et al.(supra). To generate rabbit polyclonal antibodies, and to minimize thepotential for obtaining antisera that is non-specific, or exhibitslow-affinity binding to a XAF, two or three fusions are generated foreach protein, and each fusion is injected into at least two rabbits.Antisera are raised by injections in series, preferably including atleast three booster injections.

In addition, antibodies of the invention may be produced using XAF aminoacid sequences that do reside within highly conserved regions. Forexample, amino acid sequences from the N-terminal 150 amino acids ofeither XAF-1 or XAF-2 may be used as antigen to generate antibodiesspecific toward both XAF-1 and XAF-2, and possibly specific toward othermembers of the XAF family of proteins. These antibodies may be screenedas described above.

In addition to intact monoclonal and polyclonal anti-XAF-1 antibodies,the invention features various genetically engineered antibodies,humanized antibodies, and antibody fragments, including F(ab')2, Fab',Fab, Fv and sFv fragments. Antibodies can be humanized by methods knownin the art, e.g., monoclonal antibodies with a desired bindingspecificity can be commercially humanized (Scotgene, Scotland; OxfordMolecular, Palo Alto, Calif.). Fully human antibodies, such as thoseexpressed in transgenic animals, are also features of the invention(Green et al., Nature Genetics 7: 13-21, 1994).

Ladner (U.S. Pat. No. 4,946,778 and 4,704,692) describes methods forpreparing single polypeptide chain antibodies. Ward et al. (Nature 341:544-546, 1989) describe the preparation of heavy chain variable domains,which they term "single domain antibodies," which have highantigen-binding affinities. McCafferty et al. (Nature 348: 552-554,1990) show that complete antibody V domains can be displayed on thesurface of fd bacteriophage, that the phage bind specifically toantigen, and that rare phage (one in a million) can be isolated afteraffinity chromatography. Boss et al. (U.S. Pat. No. 4,816,397) describevarious methods for producing immunoglobulins, and immunologicallyfunctional fragments thereof, which include at least the variabledomains of the heavy and light chain in a single host cell. Cabilly etal. (U.S. Pat. No. 4,816,567) describe methods for preparing chimericantibodies.

VII. Use of XAF Antibodies

Antibodies to XAF proteins may be used, as noted above, to detect XAFproteins or to inhibit the biological activities of XAF proteins. Inaddition, the antibodies may be coupled to compounds for diagnosticand/or therapeutic uses such as radionucleotides for imaging and therapyand liposomes for the targeting of compounds to a specific tissuelocation.

VIII. Detection of XAF Gene Expression

As noted, the antibodies described above may be used to monitor XAFprotein expression. In addition, in situ hybridization is a method whichmay be used to detect the expression of XAF genes. In situ hybridizationtechniques, such as fluorescent in situ hybridization (FISH), rely uponthe hybridization of a specifically labeled nucleic acid probe to thecellular RNA in individual cells or tissues. Therefore, it allows theidentification of mRNA within intact tissues, such as the heart. In thismethod, oligonucleotides or cloned nucleotide (RNA or DNA) fragmentscorresponding to unique portions of XAF genes are used to detectspecific mRNA species, e.g., in the heart. Numerous other geneexpression detection techniques are known to those of skill in the artand may be employed here.

IX. Identification of Compounds that Modulate XAF Protein Expression

Based on our experimental results, we have developed a number ofscreening procedures for identifying therapeutic compounds (e.g.,anti-apoptotic or apoptotic-inducing) which can be used in humanpatients. In particular examples, compounds that down regulateexpression of XAF proteins are considered useful in the invention fortreatment of diseases hallmarked by an excessive amount of apoptosis,such as neurodegenerative disorders. Similarly, compounds that upregulate or activate XAF proteins are also considered useful as drugsfor the treatment of diseases hallmarked by impaired apoptosis, such ascancer. In general, the screening methods of the invention involvescreening any number of compounds for therapeutically active agents byemploying any number of in vitro or in vivo experimental systems.

The methods of the invention simplify the evaluation, identification,and development of active agents for the treatment and prevention ofconditions involving an inappropriate amount of apoptosis, which may beexcessive or insufficient, depending upon the condition. These screeningmethods provide a facile means for selecting natural product extracts orcompounds of interest from a large population which are furtherevaluated and condensed to a few active and selective materials.Constituents of this pool are then purified and evaluated in the methodsof the invention to determine their anti-apoptotic or apoptotic-inducingactivities.

In general, novel drugs for the treatment of conditions involving anappropriate level of apoptosis are identified from large libraries ofboth natural product or synthetic (or semi-synthetic) extracts orchemical libraries according to methods known in the art. Those skilledin the field of drug discovery and development will understand that theprecise source of test extracts or compounds is not critical to thescreening procedure(s) of the invention. Accordingly, virtually anynumber of chemical extracts or compounds can be screened using theexemplary methods described herein. Examples of such extracts orcompounds include, but are not limited to, plant-, fungal-, prokaryotic-or animal-based extracts, fermentation broths, and synthetic compounds,as well as modification of existing compounds. Numerous methods are alsoavailable for generating random or directed synthesis (e.g.,semi-synthesis or total synthesis) of any number of chemical compounds,including, but not limited to, saccharide-, lipid-, peptide-, andnucleic acid-based compounds. Synthetic compound libraries arecommercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Inaddition, natural and synthetically produced libraries are produced, ifdesired, according to methods known in the art, e.g., by standardextraction and fractionation methods. Furthermore, if desired, anylibrary or compound is readily modified using standard chemical,physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their anti-apoptotic or apoptotic-inducingactivities should be employed whenever possible.

When a crude extract is found to have anti-apoptotic orapoptotic-inducing activities or both, further fractionation of thepositive lead extract is necessary to isolate chemical constituentsresponsible for the observed effect. Thus, the goal of the extraction,fractionation, and purification process is the careful characterizationand identification of a chemical entity within the crude extract havinganti-apoptotic or apoptotic-inducing activities. The same in vivo and invitro assays described herein for the detection of activities inmixtures of compounds can be used to purify the active component and totest derivatives thereof. Methods of fractionation and purification ofsuch heterogenous extracts are known in the art. If desired, compoundsshown to be useful agents for the treatment of pathogenicity arechemically modified according to methods known in the art. Compoundsidentified as being of therapeutic value are subsequently analyzed usingany standard animal model of degenerative disease or cancer known in theart.

Below we describe screening methods for identifying and evaluating theefficacy of a compound as an anti-apoptotic or apoptotic-inducing agent.These methods are intended to illustrate, not limit, the scope of theclaimed invention.

a) Screens for compounds affecting XAF protein expression

XAF cDNAs may be used to facilitate the identification of compounds thatincrease or decrease XAF protein expression. In one approach, candidatecompounds are added, in varying concentrations, to the culture medium ofcells expressing XAF mRNA. The XAF mRNA expression is then measured, forexample, by Northern blot analysis (Ausubel et al., supra) using a XAFDNA, or cDNA or RNA fragment, as a hybridization probe. The level of XAFmRNA expression in the presence of the candidate compound is compared tothe level of XAF mRNA expression in the absence of the candidatecompound, all other factors (e.g., cell type and culture conditions)being equal.

The effect of candidate compounds on XAF-mediated apoptosis may,instead, be measured at the level of translation by using the generalapproach described above with standard protein detection techniques,such as Western blotting or immunoprecipitation with a XAF-specificantibody (for example, the XAF-1 specific antibody described herein).

In an alternative approach to detecting compounds which regulate XAF atthe level of transcription, candidate compounds may be tested for anability to regulate a reporter gene whose expression is directed by aXAF gene promoter. For example, a cell unlikely to undergo apoptosis maybe transfected with a expression plasmid that includes a luciferasereporter gene operably linked to the XAF-1 promoter. Candidate compoundsmay then be added, in varying concentrations, to the culture medium ofthe cells. Luciferase expression levels may then be measured bysubjecting the compound-treated transfected cells to standard luciferaseassays known in the art, such as the luciferase assay system kit usedherein that is commercially available from Promega, and rapidlyassessing the level of luciferase activity on a luminometer. The levelof luciferase expression in the presence of the candidate compound iscompared to the level of luciferase expression in the absence of thecandidate compound, all other factors (e.g., cell type and cultureconditions) being equal.

Compounds that modulate the level of XAF protein expression may bepurified, or substantially purified, or may be one component of amixture of compounds such as an extract or supernatant obtained fromcells, from mammalian serum, or from growth medium in which mammaliancells have been cultured (Ausubel et al., supra). In an assay of amixture of compounds, XAF protein expression is tested againstprogressively smaller subsets of the compound pool (e.g., produced bystandard purification techniques such as HPLC or FPLC) until a singlecompound or minimal number of effective compounds is demonstrated tomodulate XAF protein expression.

b) Screens for compounds affecting XAF biological activity

Compounds may also be screened for their ability to modulate, forexample, XAF-1 apoptosis inducing activity. In this approach, the degreeof apoptosis in the presence of a candidate compound is compared to thedegree of apoptosis in its absence, under equivalent conditions. Again,the screen may begin with a pool of candidate compounds, from which oneor more useful modulator compounds are isolated in a step-wise fashion.Apoptosis activity may be measured by any standard assay, for example,those described herein.

Another method for detecting compounds that modulate theapoptosis-inducing activity of XAF has been to screen for compounds thatinteract physically with a given XAF polypeptide, e.g., XAF-1. Thesecompounds were detected by adapting yeast two-hybrid expression systemsknown in the art. These systems detected protein interactions using atranscriptional activation assay and are generally described by Gyuriset al. (Cell 75:791-803, 1993) and Field et al. (Nature 340:245-246,1989), and are commercially available from Clontech (Palo Alto, Calif.).In addition, PCT Publication WO 95/28497 describes a yeast two-hybridassay in which proteins involved in apoptosis, by virtue of theirinteraction with BCL-2, were detected. A similar method has been used toidentify proteins and other compounds that interacted with XAF-1, and isused to identify XAF-2 splice variant interactors.

A compound that promotes an increase in the expression or biologicalactivity of the XAF protein, e.g., XAF-1, is considered particularlyuseful in the invention; such a molecule may be used, for example, as atherapeutic to increase cellular levels of XAF-1 and thereby exploit theability of XAF-1 polypeptides to induce apoptosis. This would beadvantageous in the treatment of diseases involving insufficientapoptosis (e.g., cancer).

A compound that decreases XAF-1 activity (e.g., by decreasing XAF-1 geneexpression or biological activity) may also be used to increase cellularproliferation. This would be advantageous in the treatment ofdegenerative diseases, such as neurodegenerative diseases (e.g.,Alzheimer's disease, Huntington's disease) or other tissue-specificdegenerative diseases (e.g., cirrhosis of the liver, T-lymphocytedepletion in AIDS, hair loss).

Molecules that are found, by the methods described above, to effectivelymodulate XAF gene expression or polypeptide activity may be testedfurther in animal models. If they continue to function successfully inan in vivo setting, they may be used as therapeutics to either inhibitor enhance apoptosis, as appropriate.

X. Therapies

Therapies may be designed to circumvent or overcome a XAF gene defect orinadequate XAF gene expression, and thus modulate and possibly alleviateconditions involving an inappropriate amount of apoptosis. XAF-1 isexpressed in the every tissue looked at thus far. Hence, in consideringvarious therapies, it is understood that such therapies may be targetedat any tissues demonstrated to express XAF-1. In particular, therapiesto enhance XAF-1 gene expression are useful in promoting apoptosis incancerous cells. Apoptosis-inducing XAF-1 reagents may include, withoutlimitation, full length or fragment XAF-1 polypeptides, XAF-1 mRNA, orany compound which increases XAF-1 apoptosis-inducing activity.

a) Protein Therapy

Treatment or prevention of inappropriate apoptosis can be accomplishedby replacing mutant or surplus XAF protein with normal protein, bymodulating the function of mutant protein, or by delivering normal XAFprotein to the appropriate cells. It is also be possible to modify thepathophysiologic pathway (e.g., a signal transduction pathway) in whichthe protein participates in order to correct the physiological defect.

To replace a mutant protein with normal protein, or to add protein tocells which no longer express sufficient XAF, it is necessary to obtainlarge amounts of pure XAF protein from cultured cell systems which canexpress the protein. Delivery of the protein to the affected tissues(e.g., cancerous tissues) can then be accomplished using appropriatepackaging or administrating systems. Alternatively, small moleculeanalogs may be used and administered to act as XAF agonists and in thismanner produce a desired physiological effect. Methods for finding suchmolecules are provided herein.

b) Gene Therapy

Gene therapy is another potential therapeutic approach in which normalcopies of the XAF gene or nucleic acid encoding XAF antisense RNA areintroduced into selected tissues to successfully encode for normal andabundant protein or XAF antisense RNA in cells which inappropriatelyeither suppress cell death (e.g., cancerous ovarian cells) or enhancethe rate of cell death (e.g., neuronal cell death leading to disease),respectively. The gene must be delivered to those cells in a form inwhich it can be taken up and encode for sufficient protein to provideeffective function. Alternatively, in some mutants it may be possible topromote apoptosis by introducing another copy of the homologous genebearing a second mutation in that gene or to alter the mutation, or useanother gene to block any negative effect.

Transducing retroviral vectors can be used for somatic cell gene therapyespecially because of their high efficiency of infection and stableintegration and expression. The targeted cells however must be able todivide and the expression levels of normal protein should be high. Forexample, the full length XAF-1 gene, or portions thereof, can be clonedinto a retroviral vector and driven from its endogenous promoter or fromthe retroviral long terminal repeat or from a promoter specific for thetarget cell type of interest (such as neurons). Other viral vectorswhich can be used include adenovirus, adeno-associated virus, vacciniavirus, bovine papilloma virus, or a herpes virus such as Epstein-BarrVirus.

Gene transfer could also be achieved using non-viral means requiringinfection in vitro. This would include calcium phosphate, DEAE dextran,electroporation, and protoplast fusion. Liposomes may also bepotentially beneficial for delivery of DNA into a cell. Although thesemethods are available, many of these are lower efficiency.

Transplantation of normal genes into the affected cells of a patient canalso be useful therapy. In this procedure, a normal XAF gene istransferred into a cultivatable cell type, either exogenously orendogenously to the patient. These cells are then injectedserotologically into the targeted tissue(s).

Retroviral vectors, adenoviral vectors, adenovirus-associated viralvectors, or other viral vectors with the appropriate tropism for cellslikely to be involved in apoptosis (for example, epithelial cells) maybe used as a gene transfer delivery system for a therapeutic XAF geneconstruct. Numerous vectors useful for this purpose are generally known(Miller, Human Gene Therapy 15-14, 1990; Friedman, Science244:1275-1281, 1989; Eglitis and Anderson, BioTechniques 6: 608-614,1988; Tolstoshev and Anderson, Curr. Opin. Biotech. 1: 55-61, 1990;Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al., Nucl. Acid Res.and Mol. Biol. 36: 311-322, 1987; Anderson, Science 226: 401-409, 1984;Moen, Blood Cells 17: 407-416, 1991; Miller et al., Biotech. 7: 980-990,1989; Le Gal La Salle et al., Science 259: 988-990, 1993; and Johnson,Chest 107: 77S-83S, 1995). Retroviral vectors are particularly welldeveloped and have been used in clinical settings (Rosenberg et al., N.Engl. J. Med 323: 370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).Non-viral approaches may also be employed for the introduction oftherapeutic DNA into cells otherwise predicted to undergo apoptosis. Forexample, XAF may be introduced into a neuron or a T cell by lipofection(Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al.,Neurosci. Lett. 117: 259, 1990; Brigham et al., Am. J. Med.

Sci. 298: 278, 1989; Staubinger et al., Meth. Enz. 101:512, 1983 ,asialorosonucoid-polylysine conjugation (Wu et al., J. Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. Chem. 264: 16985, 1989); or, lesspreferably, micro-injection under surgical conditions (Wolff et al.,Science 247: 1465, 1990).

In another approach that may be utilized with all of the above methods,a therapeutic XAF DNA construct is preferably applied to the site of thedesired apoptosis event (for example, by injection). However, it mayalso be applied to tissue in the vicinity of the desired apoptosis eventor to a blood vessel supplying the cells (e.g., cancerous cells) desiredto undergo apoptosis.

In the constructs described, XAF cDNA expression can be directed fromany suitable promoter (e.g., the human cytomegalovirus (CMV), simianvirus 40 (SV40), or metallothionein promoters), and regulated by anyappropriate mammalian regulatory element. For example, if desired,enhancers known to preferentially direct gene expression in neuralcells, lymphocytes, or muscle cells may be used to direct XAFexpression. The enhancers used could include, without limitation, thosethat are characterized as tissue- or cell-specific in their expression.Alternatively, if a XAF genomic clone is used as a therapeutic construct(for example, following isolation by hybridization with the XAF cDNAdescribed above), regulation may be mediated by the cognate regulatorysequences or, if desired, by regulatory sequences derived from aheterologous source, including any of the promoters or regulatoryelements described above.

Antisense based strategies have employed to explore XAF gene functionand as a basis for therapeutic drug design. The principle is based onthe hypothesis that sequence-specific suppression of gene expression canbe achieved by intracellular hybridization between mRNA and acomplementary antisense species. The formation of a hybrid RNA duplexmay then interfere with the processing/transport/translation and/orstability of the target XAF mRNA. Antisense strategies may use a varietyof approaches including the use of antisense oligonucleotides andinjection of antisense RNA. For our analysis of XAF-1 gene function, weemployed the method of transfection of antisense RNA expression vectorsinto targeted cells. Antisense effects can be induced by control (sense)sequences, however, the extent of phenotypic changes are highlyvariable. Phenotypic effects induced by antisense effects are based onchanges in criteria such as protein levels, protein activitymeasurement, and target mRNA levels.

For example, XAF-1 gene therapy may also be accomplished by directadministration of antisense XAF-1 mRNA to a cell that is expected toundergo undesired apoptosis. The antisense XAF-1 mRNA may be producedand isolated by any standard technique, but is most readily produced byin vitro transcription using an antisense XAF-1 cDNA under the controlof a high efficiency promoter (e.g., the T7 promoter). Administration ofantisense XAF-1 mRNA to cells can be carried out by any of the methodsfor direct nucleic acid administration described above.

Another therapeutic approach within the invention involvesadministration of recombinant XAF polypeptide, either directly to thesite of a desired apoptosis event (for example, by injection) orsystemically (for example, by any conventional recombinant proteinadministration technique). The dosage of XAF depends on a number offactors, including the size and health of the individual patient, but,generally, between 0.1 mg and 100 mg inclusive are administered per dayto an adult in any pharmaceutically acceptable formulation.

XI. Administration of XAF Polypeptides, XAF Genes, or Modulators of XAFSynthesis or Function

A XAF protein, gene, or modulator may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form.

Conventional pharmaceutical practice may be employed to provide suitableformulations or compositions to administer neutralizing XAF antibodiesor XAF-inhibiting compounds (e.g., antisense XAF-1 or a XAF-1 dominantnegative mutant) to patients suffering from a disease (e.g., adegenerative disease) that is caused by excessive apoptosis.Administration may begin before the patient is symptomatic. Anyappropriate route of administration may be employed, for example,administration may be parenteral, intravenous, intra-arterial,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, intranasal, aerosol, by suppositories, or oraladministration. Therapeutic formulations may be in the form of liquidsolutions or suspensions; for oral administration, formulations may bein the form of tablets or capsules; and for intranasal formulations, inthe form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in Remington's Pharmaceutical Sciences, (18^(th) edition), ed.A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for XAF modulatory compounds include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel.

If desired, treatment with a XAF protein, gene, or modulatory compoundmay be combined with more traditional therapies for the diseaseinvolving excessive apoptosis, such as surgery, steroid therapy, orchemotherapy for autoimmune disease; antiviral therapy for AIDS; andtissue plasminogen activator (TPA) for ischemic injury. Likewise,treatment with a XAF protein, gene, or modulatory compound may becombined with more traditional therapies for the disease involvinginsufficient apoptosis, such as surgery, radiation therapy, andchemotherapy for cancer.

XII. Detection of Conditions Involving Altered Apoptosis

XAF polypeptides and nucleic acid sequences find diagnostic use in thedetection or monitoring of conditions involving aberrant levels ofapoptosis. For example, decreased expression of XAF-1 may be correlatedwith decreased apoptosis in humans. Accordingly, a decrease or increasein the level of XAF-1 production may provide an indication of adeleterious condition. Levels of XAF expression may be assayed by anystandard technique. For example, XAF expression in a biological sample(e.g., a biopsy) may be monitored by standard Northern blot analysis ormay be aided by PCR (see, e.g., Ausubel et al., supra; PCR Technology:Principles and Applications for DNA Amplification, H. A. Ehrlich, Ed.Stockton Press, NY; Yap et al. Nucl. Acids. Res. 19: 4294, 1991).

Alternatively, a biological sample obtained from a patient may beanalyzed for one or more mutations in XAF nucleic acid sequences using amismatch detection approach. Generally, these techniques involve PCRamplification of nucleic acid from the patient sample, followed byidentification of the mutation (i.e., mismatch) by either alteredhybridization, aberrant electrophoretic gel migration, binding orcleavage mediated by mismatch binding proteins, or direct nucleic acidsequencing.

Any of these techniques may be used to facilitate mutant XAF detection,and each is well known in the art; examples of particular techniques aredescribed, without limitation, in Orita et al. (Proc. Natl. Acad. Sci.USA 86: 2766-2770, 1989) and Sheffield et al. (Proc. Natl. Acad. Sci.USA 86: 232-236, 1989).

In yet another approach, immunoassays are used to detect or monitor XAFprotein expression in a biological sample. XAF-specific polyclonal ormonoclonal antibodies (produced as described above) may be used in anystandard immunoassay format (e.g., ELISA, Western blot, or RIA) tomeasure XAF polypeptide levels. These levels would be compared towild-type XAF levels. For example, a decrease in XAF-1 production mayindicate a condition involving insufficient apoptosis. Examples ofimmunoassays are described, e.g., in Ausubel et al., supra.Immunohistochemical techniques may also be utilized for XAF detection.For example, a tissue sample may be obtained from a patient, sectioned,and stained for the presence of XAF using an anti-XAF antibody and anystandard detection system (e.g., one which includes a secondary antibodyconjugated to horseradish peroxidase). General guidance regarding suchtechniques can be found in, e.g., Bancroft and Stevens (Theory andPractice of Histological Technigues, Churchill Livingstone, 1982) andAusubel et al. (supra).

In one preferred example, a combined diagnostic method may be employedthat begins with an evaluation of XAF protein production (for example,by immunological techniques or the protein truncation test (Hogerrorstet al., Nature Genetics 10: 208-212, 1995) and also includes a nucleicacid-based detection technique designed to identify more subtle XAFmutations (for example, point mutations). As described above, a numberof mismatch detection assays are available to those skilled in the art,and any preferred technique may be used. Mutations in XAF may bedetected that either result in loss of XAF expression or loss of normalXAF biological activity. In a variation of this combined diagnosticmethod, XAF-1 biological activity is measured as apoptotic-inducingactivity using any appropriate apoptosis assay system (for example,those described herein).

Mismatch detection assays also provide an opportunity to diagnose aXAF-mediated predisposition to diseases caused by inappropriateapoptosis. For example, a patient heterozygous for a XAF-1 mutation thatinduces a XAF-1 overexpression may show no clinical symptoms and yetpossess a higher than normal probability of developing one or more typesof neurodegenerative, myelodysplastic or having severe sequelae to anischemic event. Given this diagnosis, a patient may take precautions tominimize their exposure to adverse environmental factors (for example,UV exposure or chemical mutagens) and to carefully monitor their medicalcondition (for example, through frequent physical examinations). Thistype of XAF-1 diagnostic approach may also be used to detect XAF-1mutations in prenatal screens. The XAF-1 diagnostic assays describedabove may be carried out using any biological sample (for example, anybiopsy sample or other tissue) in which XAF-1 is normally expressed.Identification of a mutant XAF-1 gene may also be assayed using thesesources for test samples.

Alternatively, a XAF mutation, particularly as part of a diagnosis forpredisposition to XAF-associated degenerative disease, may be testedusing a DNA sample from any cell, for example, by mismatch detectiontechniques. Preferably, the DNA sample is subjected to PCR amplificationprior to analysis.

XIII. Preventative Anti-Apoptotic Therapy

In a patient diagnosed to be heterozygous for a XAF mutation or to besusceptible to XAF mutations or aberrant XAF expression (even if thosemutations or expression patterns do not yet result in XAF overexpressionor increased XAF biological activity), or a patient diagnosed with adegenerative disease (e.g., motor neuron degenerative diseases such asSMA or ALS diseases), or diagnosed as HIV positive, any of the abovetherapies may be administered before the occurrence of the diseasephenotype. For example, the therapies may be provided to a patient whois HIV positive but does not yet show a diminished T cell count or otherovert signs of AIDS. In particular, compounds shown to decrease XAF-1expression or XAF-1 biological activity may be administered to patientsdiagnosed with degenerative diseases by any standard dosage and route ofadministration (see above). Alternatively, gene therapy using aantisense XAF-1 mRNA expression construct may be undertaken to reverseor prevent the cell defect prior to the development of the degenerativedisease.

The methods of the instant invention may be used to reduce or diagnosethe disorders described herein in any mammal, for example, humans,domestic pets, or livestock. Where a non-human mammal is treated ordiagnosed, the XAF polypeptide, nucleic acid, or antibody employed ispreferably specific for that species.

XIV. Identification of Additional XAF Genes

Standard techniques, such as the polymerase chain reaction (PCR) and DNAhybridization, may be used to clone additional XAF homologues in otherspecies. Southern blots of murine genomic DNA hybridized at lowstringency with probes specific for human XAF reveal bands thatcorrespond to XAF and/or related family members. Thus, additional XAFsequences may be readily identified using low stringency hybridization.Furthermore, murine and human XAF-specific primers may be used to cloneadditional XAF related genes by RT-PCR.

Thus far, we have identified multiple ESTs in the data base that havesignificant homology to XAF-1. From the EST sequences, we have madeoligo primers and PCR cloned "XAF-2." The N terminus of the XAF-2protein has five of the amino-terminal zinc fingers of XAF-1, with aunique carboxy terminus that has two additional RING zinc fingers, sothat the entire XAF-2 protein, like XAF-1, has seven Zinc finger bindingdomains.

XV. Characterization of XAF Activity and Intracellular LocalizationStudies

The ability of XAF proteins to modulate apoptosis can be defined in invitro systems in which alterations of apoptosis can be detected.Mammalian expression constructs carrying XAF cDNAs, which are eitherfull-length or truncated, can be introduced into cell lines such as CHO,NIH 3T3, HL60, Rat-1, or Jurkat cells. In addition, SF9 insect cells maybe used, in which case the XAF gene is preferentially expressed using aninsect baculovirus expression system. Following transfection, apoptosiscan be induced by standard methods, which include serum withdrawal, orapplication of staurosporine, menadione (which induces apoptosis viafree radical formation), or anti-Fas or anti-TNF-R1 antibodies. As acontrol, cells are cultured under the same conditions as those inducedto undergo apoptosis, but either not transfected, or transfected with avector that lacks a XAF insert. The ability of each XAF construct toinduce or inhibit apoptosis upon expression can be quantified bycalculating the survival index of the cells, i.e., the ratio ofsurviving transfected cells to surviving control cells. Theseexperiments can confirm the presence of apoptosis inducing activity ofthe full length XAF-1 protein and, as discussed below, can also be usedto determine the functional region(s) of XAF-1 protein. These assays mayalso be performed in combination with the application of additionalcompounds in order to identify compounds that modulate apoptosis via XAFexpression.

XVI. Examples of Additional Apoptosis Assays

Specific examples of apoptosis assays are also provided in the followingreferences. Assays for apoptosis in lymphocytes are disclosed by: Li etal., "Induction of apoptosis in uninfected lymphocytes by HIV-1 Tatprotein", Science 268: 429-431, 1995; Gibellini et al., "Tat-expressingJurkat cells show an increased resistance to different apoptoticstimuli, including acute human immunodeficiency virus-type 1 (HIV-1)infection", Br. J. Haematol. 89: 24-33, 1995; Martin et al., "HIV-1infection of human CD4⁺ T cells in vitro. Differential induction ofapoptosis in these cells." J. Immunol. 152:330-342, 1994; Terai et al.,"Apoptosis as a mechanism of cell death in cultured T lymphoblastsacutely infected with HIV-1", J. Clin Invest. 87: 1710-1715, 199 1;Dhein et al., "Autocrine T-cell suicide mediated by APO-1/(Fas/CD95)",Nature 373: 438-441, 1995; Katsikis et al., "Fas antigen stimulationinduces marked apoptosis of T lymphocytes in human immunodeficiencyvirus-infected individuals", J. Exp. Med. 1815:2029-2036, 1995;Westendorp et al., "Sensitization of T cells to CD95-mediated apoptosisby HIV-1 Tat and gp120", Nature 375:497, 1995; DeRossi et al., Virology198:234-244, 1994.

Assays for apoptosis in fibroblasts are disclosed by: Vossbeck et al.,"Direct transforming activity of TGF-beta on rat fibroblasts", Int. J.Cancer 61:92-97, 1995; Goruppi et al., "Dissection of c-myc domainsinvolved in S phase induction of NIH3T3 fibroblasts", Oncogene9:1537-44, 1994; Fernandez et al., "Differential sensitivity of normaland Ha-ras transformed C3H mouse embryo fibroblasts to tumor necrosisfactor: induction of bcl-2, c-myc, and manganese superoxide dismutase inresistant cells", Oncogene 9:2009-2017, 1994; Harrington et al.,"c-Myc-induced apoptosis in fibroblasts is inhibited by specificcytokines", EMBO J. 13:3286-3295, 1994; Itoh et al., "A novel proteindomain required for apoptosis. Mutational analysis of human Fasantigen", J. Biol. Chem. 268:10932-10937, 1993.

Assays for apoptosis in neuronal cells are disclosed by: Melino et al.,"Tissue transglutaminase and apoptosis: sense and antisense transfectionstudies with human neuroblastoma cells", Mol. Cell Biol. 14:6584-6596,1994; Rosenbaum et al., "Evidence for hypoxia-induced, programmed celldeath of cultured neurons", Ann. Neurol. 36:864-870, 1994; Sato et al.,"Neuronal differentiation of PC12 cells as a result of prevention ofcell death by bcl-2", J. Neurobiol 25:1227-1234, 1994; Ferrari et al.,"N-acetylcysteine D- and L-stereoisomers prevents apoptotic death ofneuronal cells", J. Neurosci. 1516:2857-2866, 1995; Talley et al.,"Tumor necrosis factor alpha-induced apoptosis in human neuronal cells:protection by the antioxidant N-acetylcysteine and the genes bcl-2 andcrmA", Mol. Cell Biol. 1585:2359-2366, 1995; Talley et al., "TumorNecrosis Factor Alpha-Induced Apoptosis in Human Neuronal Cells:Protection by the Antioxidant NAcetylcysteine and the Genes bcl-2 andcrmA", Mol. Cell. Biol. 15:2359-2366, 1995; Walkinshaw et al.,"Induction of apoptosis in catecholaminergic PC 12 cells by L-DOPA.Implications for the treatment of Parkinson's disease.", J. Clin.Invest. 95:2458-2464, 1995.

Assays for apoptosis in insect cells are disclosed by: Clem et al.,"Prevention of apoptosis by a baculovirus gene during infection ofinsect cells", Science 254:1388-1390, 1991; Crook et al., "Anapoptosis-inhibiting baculovirus gene with a zinc finger-like motif", J.Virol. 67:2168-2174, 1993; Rabizadeh et al., "Expression of thebaculovirus p35 gene inhibits mammalian neural cell death", J.Neurochem. 61:2318-2321, 1993; Birnbaum et al., "An apoptosis inhibitinggene from a nuclear polyhedrosis virus encoding a polypeptide withCys/His sequence motifs", J. Virol. 68:2521-2528, 1994; Clem et al.,Mol. Cell. Biol. 14:5212-5222, 1994.

XVII. Construction of a Transgenic Animal

Characterization of XAF genes provides information that is necessary forXAF knockout animal models to be developed by homologous recombination.Preferably, the model is a mammalian animal, most preferably a mouse.Similarly, an animal model of XAF overproduction may be generated byintegrating one or more XAF sequences into the genome, according tostandard transgenic techniques.

A replacement-type targeting vector, which would be used to create aknockout model, can be constructed using an isogenic genomic clone, forexample, from a mouse strain such as 129/Sv (Stratagene Inc., LaJolla,Calif.). The targeting vector will be introduced into a suitably-derivedline of embryonic stem (ES) cells by electroporation to generate ES celllines that carry a profoundly truncated form of a XAF gene. To generatechimeric founder mice, the targeted cell lines will be injected into amouse blastula stage embryo. Heterozygous offspring will be interbred tohomozygosity. Knockout mice would provide the means, in vivo, to screenfor therapeutic compounds that modulate apoptosis via a XAF-dependentpathway. Making such mice may require use of loxP sites if there aremultiple copies of XAF genes (i.e., genes encoding XAF-1 and another XAFpolypeptide) on the chromosome (see Sauer and Henderson, Nucleic AidsRes. 17: 147-61, 1989).

The following examples are to illustrate the invention. They are notmeant to limit the invention in any way.

EXAMPLE I

cDNA and Predicted Amino Acid Sequences of Cloned Human XAF-1

Yeast 2-hybrid analysis (see U.S. Pat. No. 5,919,912 and relatedapplications) with XIAP as the `bait` protein identified a 37 kDa, RINGzinc finger protein termed XAF-1 (XIAP associated factor 1).

Methods

The plasmid pAS2-XIAP, which encodes the GAL4 DNA-binding domain fusedto full-length XIAP, was constructed by inserting the coding region offull length XIAP into the pAS2 plasmid which is commercially availablefrom Clontech. PAS2-XIAP was then used as bait (DNA-binding domainhybrid) in yeast two-hybrid screens of the human placenta cDNA librarycommercially available from Clontech. The yeast two-hybrid assay andisolation of positive clones and subsequent interaction analyses werecarried out as described (PCT Publication WO 95/28497). DNA sequence wasperformed on an Applied Biosytems model 373A automated DNA sequencer.

Results

Shown in FIG. 1 is the complete nucleotide sequence of XAF-1 cDNAdetermined for the coding strand (SEQ ID NO.:1; EMBL accession numberX99699) and is shown with its encoded protein below in single lettercode (SEQ ID NO.: 2). The asterisk indicates the stop codon. The entireXAF-1 protein is predicted to have seven Zinc finger binding domains,six of which are located in the N-terminal 178 amino acids. XAF-1displays significant homology to members of the TRAF family,particularly TRAF6, but lacks the TRAF-C and TRAF-N domains.

EXAMPLE II

Predicted Zinc Fingers of XAF-1 Amino-terminus

Results

Shown on FIG. 2 is a schematic of the six predicted Zinc finger bindingdomains corresponding to the N-terminal 178 amino acids of XAF-1 (SEQ IDNO.: 6).

EXAMPLE III

Northern Blot Analysis of XAF-1 mRNA in Multiple Human Tissues

Methods

Using methods described in the art (see, for example, Ausubel, et al.,supra), mRNA was collected from tissues from heart, brain, placenta,lunch, liver, skeletal muscle, kidney, pancreas, spleen, thymus,prostate, testis, ovary, small intestine, mucosal lining of the colon,and peripheral blood leukocytes. mRNA was also collected from thefollowing cell lines:

HL-60. a promyelocytic leukemia;

HeLa/S3, a cervix epitheliod carcinoma;

K-562, a chronic myelogenous leukemia;

MOLT-4, a lymphobastic leukemia;

Raji, a Burkitt's lymphoma;

SW480, a colorectal adenocarcinoma;

A549, a lung carcinoma; and

G361, a melanoma.

The mRNA samples were electrophoretically resolved and transferred to anitrocellulose membrane, which was then subjected to Northern blotanalysis for the presence and expression levels of XAF-1 mRNA usingradioisotope labeled XAF-1 cDNA as a probe (as described in Ausubel, etal., supra).

Additional mRNA was also collected from lung, trachea, and placenta, aswell as various subunits of the brain, heart, testis, kidney, and fetaltissue. RNA from yeast and E. Coli bacteria was also collected. ThisRNA, as well as DNA collected from human, E. Coli bacteria, and yeast,was dot-blotted on a dot-blot apparatus, electrophoretically transferredto a nitrocellulose membrane, and probed with radioisotope labeled XAF-1cDNA for the presence and expression levels of XAF-1 mRNA.

Results

mRNA encoding XAF-1 is clearly expressed in normal cells in varioustissues. FIG. 3 shows a Northern blotting analysis reveals XAF-1 mRNA tobe widely distributed among the various tissues tested, with expressionlevels highest in the heart, placenta, spleen, thymus, ovary, smallintestine, mucosal lining of the colon, and peripheral blood leukocytes.XAF-1 mRNA is also present in K-562 and MOLT-4 leukemic cell lines.

The dot-blot analysis of various tissues shown in FIG. 4 reveals thatXAF-1 mRNA is widely distributed among the various indicated regions ofthe brain, heart, testes, kidney, lung, trachea, placenta, and fetaltissue. XAF-1 mRNA is not found, however, in yeast or the E. coli strainof bacteria.

EXAMPLE IV

Genomic southern blot analysis of XAF-1

Methods

Genomic DNA was prepared from HEC38-0 human endometrial adenocarcinomacells available from the ATCC (Bethesda, Md.) and Raji cells, digestedwith BamH1, EcoR1 and HindIII restriction endonucleases,electrophoretically resolved and transferred to a nitrocellulosemembrane. Membrane bound DNA was subjected to Southern blot analysisusing radioisotope labeled XAF-1 cDNA as a probe.

Results

As shown in FIG. 5, the gene encoding XAF-1 appears to be limited incopy number in the human genome and is the same in both HEC38-0 and Rajicells, indicating that there is most likely only one gene encodingXAF-1, and that this gene is the same in the two cell lines assayed.

EXAMPLE V

Western Blot Analysis of XAF-1 Protein in Various Cell Lines

Methods

A number of transformed, immortalized and a primary cell line weretested by Western blot analysis for the presence and expression levelsof XAF-1 protein using mouse polyclonal anti-XAF-1 antisera, which wereobtained by providing GST-fusion proteins of XAF-1 and XIAP to the MBLCo., Ltd. (Japan) for use as immunogens. Cells were lysed, and lysatesSDS-PAGE resolved, electrophoretically transferred to a nylon membrane,and immunoblotted with anti-XAF-1 polyclonal antisera. Themembrane-bound proteins were then blotted with commercially availablehorseradish peroxidase conjugated anti-mouse secondary antibody andvisualized with a chemiluminescent substrate.

The cell lines used in Western blotting analysis were:

HeLa: Epitheliod carcinoma, cervix, human;

A431: Epidermoid carcinoma, human;

SUDHL6: Hodgekin's lymphoma, human;

P19: Embryonal carcinoma, mouse;

cos-7: Kidney fibroblast, SV40 transformed, African green monkey;

293T: Adenovirus type 5 transformed primary embryonal kidney, human;

CHO: Chinese hamster ovary;

For use as a positive control for Western blotting analysis, 293Tcellstransiently expressing a myc-tagged XAF-1 protein were generated by thefollowing method:

293T cells (2×10⁵) were transfected with 4 μg of plasmid DNA encodingXAF-1 by standard lipofection methods using Trans-IT lipofection reagentcommercially available from Mirus.

Results

Shown in FIG. 6 is the Western blotting analysis of the various celllines for XAF-1 expression. By this type of analysis, XAF-1 expressionappears to be ubiquitous, with low levels seen in a number oftransformed cell lines.

EXAMPLE VI

XAF-1 Constructs and Expression

Methods

Mammalian expression vectors encoding full length XAF-1, the N-terminal172 amino acids of XAF-1 containing six potential zinc fingers,including the region with significant homology to TRAF4 and TRAF6(XAF-1N; SEQ ID NO.: 7), the C-terminal 173-317 amino acids of XAF-1containing a single potential zinc finger domain (XAF-1C; SEQ ID NO.: 8)were constructed by insertion of each coding region into the pcDNA3-mycexpression vector which contains an N-terminal c-myc epitope sequence(similar vectors are commercially available from Invitrogen). Togenerate the XAF-1 antisense construct, a 720 bp fragment of XAF-1corresponding to 723-1 nucleotides (non-coding orientation) was clonedinto the pcDNA3 expression vector (Invitrogen).

293T cells (2×10⁵) were transiently transfected with 4 μg of plasmid DNAencoding XAF-1, XAF-1N, or XAF-1C by standard lipofection methods usingTrans-IT lipofection reagent commercially available from Mirus. About 48hours following transfection, the cells were lysed, and 10⁶ cellequivalents were resolved by SDS-PAGE and electrophoreticallytransferred to a nylon membrane. The membrane-bound proteins were thenimmunoblotted with an anti-myc monoclonal antibody (9E10) (commerciallyavailable from Amersham Life Sciences), followed by a commerciallyavailable horseradish peroxidase conjugated secondary anti-mouseantibody. Immunoreactive proteins were visualized by chemiluminescencefollowing addition of substrate.

Results

Shown in FIG. 7 are schematic diagrams of the polypeptides encoded forby the various XAF-1 constructs. Although XAF-1 antisense is shown herein the "coding" orientation, in the vector, it inserted and expressed inthe "non-coding" orientation.

Shown in FIG. 8 is the Western blot analysis of 293T cells transientlytransfected with XAF-1, XAF-1N and XAF-1C probed with anti-c-mycantibody. The expressed proteins show correct electrophoretic mobilitypredicted from the amino acid sequences.

EXAMPLE VII

Effect of XAF-1 Overexpression on Cell Survival.

Methods

Recombinant adenoviruses were constructed that overexpress either theLacZ protein (negative control), p53 (positive control for cell cyclearrest), or the XAF-1 protein. HeLa (cervical carcinoma, available fromthe ATCC, Manassas, Va.) and HEL (human embryonic in lung epithelialcells, available from the ATCC, Manassas, Va.) were infected withrecombinant adenovirus at a multiplicity of infection (MOI) of 10.Triplicate samples of infected cells were harvested at t=0, 24, 48, 72,and 96 hours post infection. Cell viability was assessed using standardMTT assays. Briefly, the media was removed from the well and replacedwith 1/10 volume of MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoleum bromide, availablefrom Sigma) in phosphate buffered saline and incubated at 37° C. for 4hours. Converted dye was then extracted using acidic isopropanol (0.1 NHCl in 100% isopropanol) and absorbance determined at 570 nm in aspectrophotometer. Conversion of the substrate to the 570 nm absorbingdye is carried out by mitochondrial enzymes active in living, but notdead cells.

The methods are further described in: Carmichael, J. et al., (1987)Cancer Res. 47:936-942 and Miyake, S et al., (1996) Proc. Natl. Acad.Sci. USA 93:1320-1324.

Results

As seen in FIGS. 9 and 10, adenovirus-LacZ had no effect on cellviability (compare to the control, CON, which were not infected). Incontrast, p53 induced a profound decrease in the number of viable cellswhen primary HEL cells are used (FIG. 9), but not in the HeLa cancercell line (FIG. 10). The XAF-1 expressing adenovirus resulted in asimilar decrease in the number of viable cells in both HEL and HeLa celllines. The decrease in viability in the HeLa cell lines would thereforeseem to be p53a independent. Photographs of adeno-LacZ infected,adeno-p53 infected and adeno-XAF-1 infected HEL (FIGS. 11A, 11B, 11C)and HeLa cells (FIGS. 12A, 12B, 12C) are included. The morphology of theXAF-1 overexpressing HEL cells is consistent with cell cycle arrest. Incontrast, the XAF-1 overexpressing HeLa cells demonstrate classicalfeatures of apoptosis, including pyknotic nuclei and extensive blebbing.Photographs were taken four days post-infection using a standardphase-contrast, inverted tissue culture microscope.

EXAMPLE VIII

Cell Cycle Analysis on XAF-1 Overexpressing HEL and HeLa Cells.

Methods

1×10⁵ HeLa or HEL cells were infected at an MOI of 10 with recombinantadenoviruses expressing either LacZ (negative control), p53 (positivecontrol for cell cycle arrest) or XAF-1. Cell were harvested at 96 hourspost-infection, rinsed with PBS and fixed with 100% ethanol. Fixed cellswere centrifuged 5 min at 1000 RPM, the ethanol removed, and the cellsresuspended in 1 ml PBS. 100 μl of 0.1 mg/ml RNAse was added and thecells incubated at 37° C. for 30 minutes. 100 μl of 1 mg/ml propidiumiodide was added to stain for DNA content. Cells were then analyzed on aFACS machine and cell cycle effects examined.

Results

In HEL cells, adeno-LacZ infection had no effect on the cell cycleprofiles (compare FIG. 13A [uninfected] with FIG. 13B [LacZ infected]).In contrast, both p53 (FIG. 13C) and XAF-1 (FIG. 13D) expressingadenoviruses caused a virtually complete cessation of cell cycle and aG1 arrest (note absence of S phase cells and accumulation of G1 arrestedcells). The effects of p53 and XAF-1 were identical. Infection of HeLacells with the LacZ virus had no effect, as seen in FIGS. 14A and 14B).In contrast to the HEL cells, HeLa cells did not arrest when infectedwith the adeno-p53 virus (FIG. 14C). With the adeno-XAF-1 virus, HeLacells did not arrest in G1, but instead underwent apoptosis (FIG. 14C).(Note: the changing scales on the FACS outputs give the impression of aG2 arrest [i.e., cell with 2n DNA]. In fact, the numbers of cells in Sand G2 did not change significantly). There is a loss of G1 cells and anincrease in the number of cells with less than 1n DNA content,indicating apoptosis.

EXAMPLE IX

Chromosomal Localization of the XAF-1 Gene by Fluorescent in situHybridization (FISH).

Methods

FISH was performed on freshly isolated mouse spleen lymphocytes culturedin RPMI 1640 media containing 15% fetal calf serum, 3 μg/ml concanavalinA, 10 μg/ml lipopolysaccharide, and 50 nM mercaptoethanol. Lymphocyteswere synchronized with 180 μg/ml BrdU for 14 hours followed by 4 hrgrowth in α-MEM containing 2.5 μg/ml thymidine. Chromosome spreads wereprepared on slides using hypotonic lysis, after which the chromosomeswere fixed and air dried. 1 μg of DNA probe derived from a XAF-1specific genomic phage clone was labeled with biotinylated dATP usingthe BRL BioNick labeling kit at 15° C. for 1 hr (Gibco BRL). Slides werebaked at 55° C. for 1 hr, RNAse A treated, and the chromosomes denaturedin 70% formamide in 2×SSC for 2 min at 70° C., followed by ethanoldehydration. Probe hybridization to the denatured chromosomes wasperformed overnight in 50% formamide, 10% dextran sulphate, 1 μg/mlmouse cot I DNA. Slides were washed with 2×SSC/50% formamide followed by2×SSC at 42° C. Biotin labeled DNA was amplified and detected usingfluorescein isothiocyanate conjugated avidin and anti-avidin antibodies(FIG. 15A). Chromosomes were counterstained with Giemsa and photographed(FIG. 15B).

Results

The XAF-1 gene was found to map to the extreme end of chromosome 17, inthe p13.3 region. This region is known to encode an as yet unidentifiedtumor suppressor gene(s). This tumor suppressor gene is believed to beinvolved in a large number of tumor types, including uterine cervicalcarcinoma (Park et al., Cancer Genet. Cytogenet. 79: 74-78, 1995),breast tumors (Cornelis et al., Cancer Res. 54: 4200-4206, 1994, Merloet al., Cancer Genet. Cytogenet. 76: 106-111, 1994), gastric carcinoma(Kim et al., Lab. Invest. 72: 232-236, 1995), ovarian epithelial cancer(Wertheim et al., Oncogene 12: 2147-2153, 1996), pediatricmedulloblastoma (McDonald et al., Genomics 23: 229-232, 1994, reviewedin Cogan and McDonald, J. of Neuro-Oncology 29: 103-112, 1996) and lungcarcinoma (White et al., Br. J. Cancer 74: 863-870, 1996). Thus XAF-1maybe a tumor suppressor and therapies designed to over-express XAF-1 incancer cells may be effective (i.e., gene therapy, compounds thatup-regulate endogenous XAF-1 or compounds that activate the XAF-1pathway). Furthermore, the XAF-1 gene may provide an impolrtantstaging/prognostic indicator in cancer diagnostics through thedevelopment of a LOH type assay using PCR based detection ofmicrosatellites in the XAF-1 locus.

EXAMPLE X

Sub-cellular Localization of the XAF-1 Protein.

Methods

Triplicate plates of HeLa cells (ATCC, Bethesda, Md.) were infected witha recombinant adenovirus expressing the XAF-1 open reading frame underthe control of the chicken β-actin promoter at a multiplicity ofinfection 10. At 48 hrs post infection, the cells were harvested in 5 mlof phosphate buffered saline, pelleted by low speed centrifugation (5min, 1000 rpm in a Beckman JA-10 rotor at 4° C.), and cell extractsprepared as follows:

cells were washed with isotonic Tris buffered saline (pH 7.0)

cells were lysed by freeze/thawing 5 times in Cell Extraction Buffer (50mM PIPES, 50 mM KCI, 5 mM EGTA, 2 mM MgCl₂, 1 mM DTT, and 20 μMcytochalasin B)

nuclei were pelleted by centrifugation at 5000 RPM in a JA-17 rotor for5 minutes. Nuclear pellet was resuspended in isotonic Tris pH7.0, andfrozen at -80° C.

cytoplasmic extract was further processed by centrifugation at 60,000RPM in a TA 100.3 rotor for 30 minutes. Supernatant (cytoplasmicextract) was frozen at -80° C. Pelleted material (membrane fraction) wasresuspended in isotonic Tris pH 7.0, and frozen.

nuclear, membrane, and cytoplasmic fractions were electrophoresed on a12.5% SDS polyacrylamide gel, and electroblotted onto PVDF membranes.

Western blotting was first performed using rabbit polyclonal anti-XAF-1antibody at a concentration of 1:1,500 in Tris buffered salinecontaining 0.5% NP-40 and 3% skim milk powder. The secondary antibodywas a horse radish peroxidase coupled goat anti-rabbit IgG (Amersham)used at 1:2000 dilution in the same buffer system. Chemiluminescentdetection of bound antibody was performed using Amersham's ECL kitaccording to the manufacturer's directions. The membrane was thenre-probed with polyclonal anti-XIAP antibody at 1:2000 dilution andprocessed as above.

Results

FIG. 16A demonstrates that the vast majority of the adenovirus expressedXAF-1 protein fractionates in the nuclear compartment. A very smallfraction of the protein was observed in the membrane fraction, likely asa result of incomplete separation of the nuclear and membrane fractions.None of the protein was observed in the cytoplasmic fraction. FIG. 16Bdemonstrates that overexpression of the XAF-1 protein resulted in are-distribution of >1/2 of the endogenous XIAP protein from thecytoplasmic fraction to the nuclear fraction. One explanation for thisis that the function of XAF-1 is to relocate the XIAP protein to its`real` site of action, in the nucleus. Alternatively, XIAP may beinterfering with the function of XAF-1 in the nucleus.

EXAMPLE XI

XAF-1 Protein is Found in the Nucleus by GFP Staining.

Methods

An expression vector called pGFP-XAF-1 was constructed that generates afusion protein between green fluorescent protein (GFP) and XAF-1(Clontech). The coding region of GFP was fused to the amino terminus ofthe full length XAF-1 coding region. CHO-K1 cells or 3Y1 primary ratembryo fibroblast cells from Fischer rat fetus (available from the Rikengene bank, Tsukuba, Japan) were transiently transfected by standardlipofection methods using the Trans-IT lipofection reagent commerciallyavailable from Mirus with pGFP or pGFP-XAF-1. 24 hours followingtransfection, the cells were visualized on a fluorescent microscope witha blue filter.

All cells were counter stained with evans blue.

Results

FIGS. 17A, 17B, and 17C are photographs of transfected CHO-K1 cells.FIGS. 17A and 17B shows that in CHO-K1 cells transiently transfectedwith pGFP-XAF-1, the GFP-labeled XAF-1 protein was localized to thenucleus. This is in contrast to the GFP homogenously distributedthroughout the cytoplasm and nucleus in the CHO-K1 cells transientlytransfected with pGFP shown on FIG. 17C.

FIGS. 18A and 18B shows the GFP homogenously distributed throughout thecytoplasm and nucleus in 3Y1 cells transiently transfected with pGFP.

FIGS. 19A and 19B shows the GFP-labeled XAF-1 protein localized to thenucleus in 3Y1 cells transiently transfected with pGFP-XAF-1.

We have furthermore found that XAF-1 expression resulted in are-distribution of XIAP protein from the cytoplasm to the nucleus.

EXAMPLE XII

Neither XAF-1 nor Mammalian IAPs Over-expression can Induce NF-κBActivation in 293 T Cells.

The members of the growing family of TRAF proteins each possesses anamino terminal RING zinc finger and/or additional zinc fingers, aleucine zipper, and a unique, conserved carboxy terminal coiled coilmotif, the TRAF-C domain, which defines the family. TRAF1 and TRAF2 werefirst identified as components of the TNF-R2 signaling complex (Rothe etal., Cell 78: 681-692, 1994). The interaction of the TRAF proteins arecomplex, reflecting their putative role as adapter molecules thatexhibit no apparent enzymatic activity themselves.

Methods

Mammalian expression vectors encoding XAF-1, HIAP-1, HIAP-2, XIAP,TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, RIP, and TRADD wereconstructed by insertion of each coding region into the pcDNA3-mycexpression vector which contains an N-terminal c-myc epitope sequence(similar vectors are commercially available from Invitrogen). The NF-κBfirefly luciferase reporter plasmid pELAM-Lu was constructed byinsertion of PCR-amplified E-selectin promoter sequences from position-730 to position 52 into the pGL3-Basic vector which is commerciallyavailable from Promega.

293T cells were seeded into collagen-coated six-well plates at 2×10⁵cells per well 24 hrs before transfection. Cells were then transfectedwith 0.5 μg of pELAM-Lu reporter plasmid, 0.05 μg of pRL-CMV, 1 μg ofindicated expression plasmid and enough pCMV-myc control plasmid to give4 μg of total DNA by standard lipofection methods using Trans-ITlipofection reagent commercially available from Mirus. Twenty-four hoursafter transfection, cells were washed with PBS and lysed in 400 μl ofPassive Lysis Buffer commercially available from Promega. Lysate (20 μl)from each samples was used to measure firefly luciferase activity.Firefly luciferase activity was determined and normalized on the basisof Renilla luciferase expression level. Luciferase activity was measuredin a model TD20/20 luminometer using the Dual luciferase assay systemaccording to the manufacture's protocol (Promega). Values shown areaverages for an experiment in which each transfection was performed induplicate.

Results

XAF-1, HIAP-1, HIAP-2, and XIAP do not induced NF-κB activation in 293 Tcells. As shown in FIG. 20, when expressed singly in 293T cells, none ofthe IAPs or XAF-1 resulted in measurable activation of NF-κB, asmeasured by luciferase activity. TRAF2, TRAF5, TRAF6, RIP, and TRADDexpression plasmids, however, all strongly transactivated the reportergene. TRAF1, TRAF3, and TRAF4 failed to transactivate the reporter.

We have also obtained data showing that XIAP can activate NF-κB in HeLacells.

EXAMPLE XIII

Co-expression of XAF-1 and Mammalian IAPs do not Induce NF-κB Activationin 293 T Cells.

Methods

293T cells were seeded into collagen-coated six-well plates at 2×10⁵cells per well 24 hrs before transfection. Cells were then transfectedwith 0.5μg of pELAM-Lu reporter plasmid, 0.05 μg of pRL-CMV, 4 μg ofindicated expression plasmid(s) and enough pCMV-myc control plasmid togive 5 μg of total DNA by standard lipofection methods using Trans-ITlipofection reagent commercially available from Mirus. Twenty-four hoursafter transfection, cells were washed with PBS and lysed in 400 μl ofPassive Lysis Buffer commercially available from Promega. Lysate (20 μl)from each samples was used to measure firefly luciferase activity.Firefly luciferase activity was determined and normalized on the basisof Renilla luciferase expression level. Luciferase activity was measuredin a model TD20/20 luminometer using the Dual luciferase assay systemaccording to the manufacture's protocol (Promega). Values shown areaverages for an experiment in which each transfection was performed induplicate.

Results

As shown in FIG. 21, none of the IAPs, alone, or in combination withXAF-1, resulted in measurable activation of NF-κB when expressed in 293Tcells. Expression of TRAF6, shown here as a positive control, did induceNF-κB activation.

EXAMPLE XIV

Dose Response Effect of XAF-1 Expression on TRAF6-mediated NF-κBActivation

Methods

293T cells were seeded into collagen-coated six-well plates at 2×10⁵cells per well 24 hrs before transfection. Cells were then transfectedwith 0.5 μg of pELAM-Lu reporter plasmid, 0.1 μg of pRL-CMV, 0.5 μg ofpCMV-TRAF6, indicated amounts of pCMV-XAF-1 and enough pCMV-myc controlplasmid to give 4 μg of total DNA by standard lipofection methods usingTrans-IT lipofection reagent commercially available from Mirus.Twenty-four hours after transfection, cells were washed with PBS andlysed in 400 μl of Passive Lysis Buffer commercially available fromPromega. Lysate (20 μl) from each samples was used to measure fireflyluciferase activity. Firefly luciferase activity was determined andnormalized on the basis of Renilla luciferase expression level.Luciferase activity was measured in a model TD20/20 luminometer usingthe Dual luciferase assay system according to the manufacture's protocol(Promega). Values shown are averages for an experiment in which eachtransfection was performed in duplicate.

Results

As the results shown in FIG. 22 demonstrate, although expression ofTRAF6 was by itself capable of inducing NF-κB activity, co-expression ofTRAF6 with XAF-1 resulted in an increased level of NF-κB activationwhich increased as the amount of XAF-1 expression increased. Hence,XAF-1 was able to enhance the NF-κB inducing abilities of TRAF6.

EXAMPLE XV

Dose Response Effect of XIAP Expression on TRAF6-mediated NF-κBActivation

Methods

293T cells were seeded into collagen-coated six-well plates at 2×10⁵cells per well 24 hrs before transfection. Cells were then transfectedwith 0.5 μg of pELAM-Lu reporter plasmid, 0.1 μg of pRL-CMV, 0.5 μg ofpCMV-TRAF6, indicated amounts of pCMV-XIAP and enough pCMV-myc controlplasmid to give 4 μg of total DNA by standard lipofection methods usingTrans-IT lipofection reagent commercially available from Mirus.Twenty-four hours after transfection, cells were washed with PBS andlysed in 400 μl of Passive Lysis Buffer commercially available fromPromega. Lysate (20 μl) from each sample was used to measure fireflyluciferase activity. Firefly luciferase activity was determined andnormalized on the basis of Renilla luciferase expression level.Luciferase activity was measured in a model TD20/20 luminometer usingthe Dual luciferase assay system according to the manufacture's protocol(Promega). Values shown are averages for an experiment in which eachtransfection was performed in duplicate.

Results

The results shown in FIG. 23 demonstrate that although expression ofTRAF6 was by itself capable of inducing NF-κB activity, co-expression ofTRAF6 with XIAP resulted in an increased level of NF-κB activation whichincreased as the amount of XIAP expression increased. Hence, XIAP wasable to enhance the NF-κB inducing abilities of TRAF6.

EXAMPLE XVI

Synergistic Effect of XAF-1 and XIAP Expression on TRAF6- andTRAF2-mediated NF-κB Activation

Methods

293T cells were seeded into collagen-coated six-well plates at 2×10⁵cells per well 24 hrs before transfection. Cells were then transfectedwith 0.5 μg of pELAM-Lu (pGL3-E-selectin promoter) and 0.05 μg ofpRL-CMV, 1 μg of pCMV-TRAF6 or 1 μg of pCMV-TRAF2, 1 μg of pCMV-XAF-1and/or pCMV-XIAP, and enough pCMV-myc control plasmid to give 4 μg oftotal DNA by standard lipofection methods using Trans-IT lipofectionreagent (Mirus). Twenty-four hours after transfection, cells were washedwith PBS and lysed in 400 μl of Passive Lysis Buffer (Promega). Lysate(20 μl) from each samples was used to measure firefly luciferaseactivity. Firefly luciferase activity was determined and normalized onthe basis of Renilla luciferase expression level. Luciferase activitywas measured in a model TD20/20 luminometer (Promega) using Dualluciferase assay system according to the manufacture's protocol(Promega). Values shown are averages for an experiment in which eachtransfection was performed in duplicate.

Results

XIAP and XAF-1 were additive in their effects on TRAF6 mediated NF-κBtransactivation, as shown on FIG. 24. FIG. 25 indicates that XIAP andXAF-1 were also able to assist in TRAF2 mediated NF-κB transactivation,although to a lesser extent than their assistance in TRAF6 mediatedNF-κB transactivation. Hence, XIAP and XAF-1 work synergistically intheir signal transducing capabilities.

EXAMPLE XVII

C-terminus of XAF-1 Enhances TRAF6-mediated NF-κB Activation

Methods

Expression plasmids that express either the amino terminal domain ofXAF-1 containing six potential zinc fingers, including the region withsignificant homology to TRAF4 and TRAF6 (XAF-1N) or the carboxy terminuscontaining a single potential zinc finger domain (XAF-1C) were testedfor their capacity to augment TRAF6 mediated NF-κB activity

293T cells (2×10⁵) were transfected with 0.5 μg of pELAM-Lu reporterplasmid, 0.1 μg of pRL-TK commercially available from Promega, 0.5 μg ofpCMV-TRAF6, 1 μg of indicated expression plasmid and enough pCMV-myccontrol plasmid to give 4 μg of total DNA. Firefly luciferase activitywere determined 24 hrs after transfection and normalized on the basis ofRenilla luciferase expression level. Values shown are averages for anexperiment in which each transfection was performed in duplicate.

Results

As FIG. 26 demonstrates, we have found that the carboxy terminus ofXAF-1 protein mediates the additive effect of XAF-1 on TRAF6 inductionof NF-κB. XAF-1N expression did not augment the ability to TRAF6 toinduce NF-κB, whereas XAF-1C augmented NF-κB induction by TRAF6substantially. Full length XAF-1, as we showed previously in FIG. 21,clearly enhanced TRAF6 induction of NF-κB.

EXAMPLE XVIII

Inhibitory Effect of Antisense XAF-1 Expression on TRAF5-andTRAF6-mediated NF-κB Activation in 293 T Cells.

Methods

To generate the bcl-2 antisense construct, a 1.5 kb EcoRI fragment ofbcl-2 was cloned in a non-coding orientation into the pcDNA3 plasmidcommercially available from Invitrogen.

293T cells (2×10⁵) were transfected with 0.5 μg of pELAM-Lu reporterplasmid, 0.1 μg of pRL-TK commercially available from Promega, 0.5 μg ofpCMV-TRAF5 or pCMV-TRAF6, 3 μg of indicated antisense plasmid: antisenseXAF-1 (240-1) or antisense bcl-2 (450-23), and enough pCMV-myc controlplasmid to give 5 μg of total DNA. Firefly luciferase activity weredetermined 24 hrs after transfection and normalized on the basis ofRenilla luciferase expression level. Values shown are averages for anexperiment in which each transfection was performed in duplicate.

Results

FIG. 27 demonstrates that expression of antisense XAF-1 significantlyinhibited TRAF6 induced activation of NF-κB and, to a lesser extent,TRAF5 induced activation of NF-κB. This inhibition was specific to XAF-1since antisense bcl-2 did not have the same effect.

EXAMPLE XIX

Inhibitory Effect of Antisense XAF-1 Expression on IL-1β-induced NF-κBActivation

Methods

293T cells (2×10⁵) were transfected with 0.5 μg of pELAM-Lu reporterplasmid, 0.1 μg of pRL-TK commercially available from Promega, indicatedamounts of antisense plasmid: antisense XAF-1 (240-1) or antisense bcl-2(1486-23), and enough pCMV-myc control plasmid to give 5 μg of totalDNA. 24 hrs after transfection, cells were treated for 6 hrs with 20ng/ml of interleukin-1β (IL-β). Firefly luciferase activity weredetermined after IL-1β treatment and normalized on the basis of Renillaluciferase expression level. Values shown are averages for an experimentin which each transfection was performed in duplicate.

Results

As shown on FIG. 28, expression of antisense XAF-1 inhibitedinterleukin-1β induced activation of NF-κB. This inhibition was specificto XAF-1 since antisense bcl-2 does not have the same effect.

EXAMPLE XX

Dose Response Effect of XAF-1 Expression on IL-1β-induced NF-κBActivation

Methods

293T cells (2×10⁵) were transfected with 0.5 μg of pELAM-Lu reporterplasmid, 0.1 μg of pRL-TK commercially available from Promega, indicatedamounts of pCMV-XAF-1 and enough pCMV-myc control plasmid to give 5 μgof total DNA. 24 hrs after transfection, cells were treated for 6 hrswith 20 ng/ml of interleukin-1β(IL-1β). Firefly luciferase activity weredetermined after IL-1β treatment and normalized on the basis of Renillaluciferase expression level. Values shown are averages for an experimentin which each transfection was performed in duplicate.

Results

Expression of full length XAF-1 augmented interleukin-1β mediatedinduction of NF-κB in a dose-dependent manner, as is demonstrated inFIG. 29.

EXAMPLE XXI

Inhibitory Effect of A20 Expression on TRAF2-, TRAF5- and TRAF6-mediatedNF-κB Activation

The A20 protein is induced by NF-κB and binds to both TRAF1 and TRAF2,again via the TRAF-C domain. Binding of A20 to TRAF2 interferes withNF-κB activation in a negative feed-back loop (Song et al., Proc. Natl.Acad. Sci. USA 93: 6721-6725,1996). It has previously been establishedthat over-expression of A20 can render cells resistant to the apoptoticeffects of TNFα (Opipari et al., J. Biol. Chem. 267: 12424-12427, 1992),and may also participate in rendering B cells resistant to apoptosisfollowing CD40 signaling (Sarma et al., 270: 12353-12346, 1995).

Methods

293T cells (2×10⁵) were transfected with 0.5 μg of pELAM-Lu reporterplasmid, 0.1 μg of pRL-TK commercially available from Promega, 0.5 μg ofpCMV-TRAF2, pCMV-TRAF5 or pCMV-TRAF6, 0.3 μg of pCMV-A20 and enoughpCMV-myc control plasmid to give 4 μg of total DNA. Firefly luciferaseactivity were determined 24 hrs after transfection and normalized on thebasis of Renilla luciferase expression level. Values shown are averagesfor an experiment in which each transfection was performed in duplicate

Results

In the experiments shown on FIG. 30, co-transfection of an A20expression vector with either TRAF2, TRAF5 or TRAF6 resulted invirtually complete inhibition of NF-κB transactivation.

EXAMPLE XXII

XAF-1 Counters the Effect of A20 Expression on TRAF6 Mediated Inductionof NF-κB

Methods

293T cells (2×10⁵) were transfected with 0.5 μg of pELAM-Lu reporterplasmid, 0.1 μg of pRL-TK commercially available from Promega, 0.5 μg ofpCMV-TRAF6, 2 μg of pCMV-XAF-1, indicated amounts of pCMV-A20 and enoughpCMV-myc control plasmid to give 5 μg of total DNA. Firefly luciferaseactivity were determined 24 hrs after transfection and normalized on thebasis of Renilla luciferase expression level. Values shown are averagesfor an experiment in which each transfection was performed in duplicate.

Results

As shown in FIG. 31, XAF-1 expression had a partial neutralizing effecton the A20-mediated inhibitory function of TRAF6-mediated NF-κBactivation.

EXAMPLE XXIII

Interaction of XAF-1 with the Various TRAFs and Mammalian IAPs

Methods

XIAP and XAF-1 coding regions were cloned in frame into the pGEX-4T-1expression vector which is commercially available from Pharmacia.Expression and purification of GST-fusion proteins were performedessentially according to the manufacturer's protocol (Pharmacia).

293T cells were transiently transfected with myc-epitope tagged TRAFsand mammalian IAPs expression vectors (5 μg). After 36 hrs, cells werelysed and cell lysates were incubated with GST-XAF-1 fusion protein orGST-control protein (Glutathione-s-transferase from SchistosomaJaponicum) immobilized on 10 μl of glutathione beads. Protein adsorbedto beads were analyzed by SDS-PAGE, followed by Western blotting usinganti-c-myc monoclonal antibody (9E10). Lanes were loaded as follows:

lane 1: HIAP-2,

lane 2: TRAF1,

lane 3: TRAF2,

lane 4: TRAF3,

lane 5: A20.

Proteins in A lanes were affinity-purified with the GST-XAF-1 fusionprotein.

Proteins in B lanes were affinity-purified with the GST-control protein.

Results

GST interaction analysis indicated that XAF-1 can form complexes with avariety of cellular proteins, including HIAP-2, TRAF1, TRAF2, and A20,as is shown on FIG. 32. In this type of analysis, indirect interactionscannot be distinguished from direct binding. For instance, XAF-1 maybind TRAF2 directly (as shown by two-hybrid analysis) which in turn caninteract with either TRAF1 or A20.

EXAMPLE XXIV

In Vitro Translated TRAF2 and HIAP-1 bind XAF-1

Methods

³⁵ S-labeled in vitro translated proteins were generated by using thevarious TRAF2 and HIAP-1 expression constructs in pcDNA3-myc with theTNT T7 Coupled Reticulocyte Lysate System, according to themanufacturer's descriptions (Promega) and ³⁵ S labeled methionine,commercially available from DuPont/NEN.

³⁵ S-labeled in vitro translated proteins were incubated with GST-XAF-1fusion protein or GST-control protein immobilized on 10 μl ofglutathione beads. Protein adsorbed to beads were analyzed by SDS-PAGE.The protein bearing gel was then dried, and adsorbed proteins weredetected by autoradiograph of the gel. The lanes were loaded as follows:

lane 1: HIAP1,

lane 2: TRAF2.

Proteins in A lanes were affinity-purified with the GST-XAF-1 fusionprotein.

Proteins in B lanes were affinity-purified with the GST-control protein.

Results

As shown on FIG. 33, both in vitro translated HIAP-1 and TRAF2 bound theGST-XAF-1 fusion protein, but do not bind the GST control protein. Sincethis experiment was done in a cell-free system, we have demonstratedthat the HIAP-1:XAF-1 and the TRAF2:XAF-1 interactions are direct.

EXAMPLE XXV

XAF-1 Directly Interacts with XIAP, HIAP-1, HIAP-2, and TRAF2

Methods

The plasmids pAS2-XIAP, pAS2-HIAP-1, pAS2-HIAP-2, pAS2-TRAF2,pAS2-TRAF4, pAS2-XAF-1, and pAS2 (vector only) which encode the GAL4DNA-binding domains fused to indicated full-length proteins, were usedas baits (DNA-binding domain hybrids) in two-hybrid screens of pGAD GHplasmids (commercially available from Clontech) encoding XIAP, HIAP-1,HIAP-2, TRAF2, TRAF4, and XAF-1 as preys (activation domain hybrids).The yeast two-hybrid assay and isolation of positive clones andsubsequent interaction analyses were carried out as described elsewhere(PCT Publication WO 95/28497). DNA sequence was performed on an AppliedBiosytems model 373A automated DNA sequencer.

Results

Shown in FIG. 34 is a listing of the XAF-1 interactions with mammalianIAPs and TRAFS found in the yeast two-hybrid assay. Our resultsindicated that XAF-1 directly interacts with XIAP, HIAP-1, HIAP-2, andTRAF2 (but not TRAF4). As has been established in the literature, TRAF2can interact with TRAF1 or A20. Since we have shown here in yeasttwo-hybrid analysis that XAF-1 binds TRAF2 directly, it may be throughthis interaction that XAF-1 is able to form a complex with TRAF1 andA20, as we showed in FIG. 32.

EXAMPLE XXVI

Identification and Cloning of Human XAF-2

Methods

We screened the database for ESTs that have significant homology toXAF-1. A number of such ESTs were identified. From the EST sequences, wehave made oligonucleotide primers and PCR cloned a cDNA encoding aprotein which we have named "XAF-2."

Results

FIG. 35 shows the partial 5' nucleic acid (SEQ ID NO.: 3) and N-terminalamino acid (SEQ ID NO.: 4) sequences of the long splice variant ofXAF-2. The N-terminus of XAF-2 protein has five zinc fingers in theN-terminal 150 amino acids which show 38% amino acid identity to XAF-1(SEQ ID NO.: 2). XAF-2 also has a unique C-terminus that has two RINGzinc fingers, so that the entire XAF-2 protein, like XAF-1, has sevenzinc finger binding domains. FIG. 36 shows sequence of the the 3'untranslated region (UTR) located approximately 250 nucleic acidresidues C-terminally to the nucleic acid sequence of FIG. 35. There areat least two splice variants of XAF-2. FIG. 37A shows the full length 5'nucleotide (above; SEQ ID NO.: 9) and amino acid (below; SEQ ID NO.: 10)sequences of the long (XAF-2L) splice variant of XAF-2. The shortersplice form of XAF-2 (XAF-2S) is spliced as indicated in FIG. 37A, withthe nucleic acid encoding XAF-2S indicated in FIG. 37B, lower sequence(SEQ ID NO.: 11). FIGS. 38A, 38B, and 38C show the indicated zinc fingerbinding domains in the amino acid sequence listings of XAF-1, XAF-2L,and XAF-2S, respectively. XAF-2L and XAF-1 shown an overall amino acidsequence identity of 27%, although the first 135 amino acids of XAF-2Land the first 131 amino acids of XAF-1 share a 40% amino acid sequenceidentity (FIG. 39). As indicated in FIG. 40, the alignment of the zincfinger binding domains in XAF-1 and XAF-2L is not equivalent: the sixthzinc domain of XAF-2L aligns with the seventh zinc domain of XAF-1.However, the two XAF molecules both have seven zinc finger bindingdomains overall.

EXAMPLE XXVII

A Screen for Candidate Compounds Which Modulate XAF-1 Expression

Compounds are screened for an ability to modulate XAF-1 expression bylooking at the ability of the compounds to modulate the expression of aluciferase reporter gene operably linked to the XAF-1 promoter.

Methods

The XAF-1 promoter firefly luciferase reporter plasmid pXAF-1prom-Lu isconstructed by insertion of PCR-amplified XAF-1 promoter sequences intoa vector such as the pGL3-Basic vector which is commercially availablefrom Promega.

COS cells are seeded into six-well plates at 2×10⁵ cells per well 24 hrsbefore transfection. Cells are then transfected with 1.0 μg ofpXAF-1prom-Lu reporter plasmid, and 3.0 μg pCMV-myc control plasmid bystandard lipofection methods using Trans-IT lipofection reagentcommercially available from Mirus. Twenty-four hours after transfection,varying concentrations of different compounds are added to the culturesupernatant of transfected cells, such that there is one compound, orcombination thereof, per well. Twelve hours following treatment with thecompound, the cells are washed with PBS and lysed in 400 μl of PassiveLysis Buffer commercially available from Promega. Lysate (20 μl) fromeach samples is used to measure firefly luciferase activity. Fireflyluciferase activity is determined and normalized on the basis of Renillaluciferase expression level. Luciferase activity is measured in a modelTD20/20 luminometer using the Dual luciferase assay system according tothe manufacture's protocol (Promega).

Results

Compound-treated cells which show an increased firefly luciferaseactivity as compared to untreated control cells indicate a compound withan ability to increase XAF-1 activity. Compound-treated cells which showa decreased firefly luciferase activity as compared to untreated controlcells indicate a compound with an ability to decrease XAF-1 activity.

Other Embodiments

In other embodiments, the invention includes any protein which issubstantially identical to a mammalian XAF polypeptide provided in FIG.1 (SEQ. ID NO.: 2), FIG. 35 (SEQ ID NO.: 4), FIG. 37A (SEQ ID NO.: 10)and FIG. 38C (SEQ ID NO.: 12); such homologues include othersubstantially pure naturally-occurring mammalian XAF proteins as well assplice variants, allelic variants; natural mutants; induced mutants; DNAsequences which encode proteins and also hybridize to the XAF DNAsequences of FIG. 1 (SEQ ID NO.: 1), FIG. 35 (SEQ ID NO.: 3), FIG. 37A(SEQ ID NO.: 9) and FIG. 37B (SEQ ID NO.: 11) under high stringencyconditions (e.g., hybridizing at 2×SSC at 40° C. with a probe length ofat least 40 nucleotides) or, less preferably, under low stringencyconditions (e.g., hybridizing at 5×SSC at 25° C. with a probe length ofat least 80 nucleotides); and proteins specifically bound by antiseradirected to a XAF polypeptide. The term also includes chimericpolypeptides that include a portion derived from a XAF polypeptide.

The invention further includes analogs of any naturally-occurring XAFpolypeptides. Analogs can differ from the naturally-occurring XAFproteins by amino acid sequence differences, by post-translationalmodifications, or by both. Analogs of the invention will generallyexhibit at least 85%, more preferably 90%, and most preferably 95% oreven 99% identity with all or part of a naturally occurring XAF-1, XAF-2N-terminus, XAF-2L, or XAF-2S amino acid sequence. The length ofsequence comparison is at least 15 amino acid residues, preferably atleast 25 amino acid residues, and more preferably more than 35 aminoacid residues. Modifications include in vivo and in vitro chemicalderivatization of polypeptides, e.g., acetylation, carboxylation,phosphorylation, or glycosylation; such modifications may occur duringpolypeptide synthesis or processing or following treatment with isolatedmodifying enzymes. Analogs can also differ from the naturally-occurringXAF-1, XAF-2 N-terminus, XAF-2L or XAF-2S polypeptide by alterations inprimary sequence. These include genetic variants, both natural andinduced (for example, resulting from random mutagenesis by irradiationor exposure to ethanemethylsulfate or by site-specific mutagenesis asdescribed in Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra).Also included are cyclized peptides, molecules, and analogs whichcontain residues other than L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., B or Y aminoacids. In addition to full-length polypeptides, the invention alsoincludes XAF-1, XAF-2 N-terminus, XAF-2L and XAF-2S polypeptidefragments. As used herein, the term "fragment," means at least 20contiguous amino acids, preferably at least 30 contiguous amino acids,more preferably at least 50 contiguous amino acids, and most preferablyat least 60 to 80 or more contiguous amino acids. Fragments of XAF-1,XAF-2 N-terminus, XAF-2L and XAF-2S polypeptides can be generated bymethods known to those skilled in the art or may result from normalprotein processing (e.g., removal of amino acids from the nascentpolypeptide that are not required for biological activity or removal ofamino acids by alternative mRNA splicing or alternative proteinprocessing events).

Preferable fragments or analogs according to the invention are thosewhich facilitate specific detection of a XAF-1, XAF-2 N terminus, XAF-2Lor XAF-2S nucleic acid or amino acid sequence in a sample to bediagnosed. Particularly useful XAF-1 fragments for this purpose include,without limitation, the amino acid fragments shown in FIG. 7.

All publications and patent applications mentioned in this specificationarc herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - <160> NUMBER OF SEQ ID NOS: 143                                      - - <210> SEQ ID NO 1                                                        <211> LENGTH: 1326                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 1                                                         - - atggaaggag acttctcggt gtgcaggaac tgtaaaagac atgtagtctc tg -             #ccaacttc     60                                                                 - - accctccatg aggcttactg cctgcggttc ctggtcctgt gtccggagtg tg -            #aggagcct    120                                                                 - - gtccccaagg aaaccatgga ggagcactgc aagcttgagc accagcaggt tg -            #ggtgtacg    180                                                                 - - atgtgtcagc agagcatgca gaagtcctcg ctggagtttc ataaggccaa tg -            #agtgccag    240                                                                 - - gagcgccctg ttgagtgtaa gttctgcaaa ctggacatgc agctcagcaa gc -            #tggagctc    300                                                                 - - cacgagtcct actgtggcag ccggacagag ctctgccaag gctgtggcca gt -            #tcatcatg    360                                                                 - - caccgcatgc tcgcccagca cagagatgtc tgtcggagtg aacaggccca gc -            #tcgggaaa    420                                                                 - - ggggaaagaa tttcagctcc tgaaagggaa atctactgtc attattgcaa cc -            #aaatgatt    480                                                                 - - ccagaaaata agtatttcca ccatatgggt aaatgttgtc cagactcaga gt -            #ttaagaaa    540                                                                 - - cactttcctg ttggaaatcc agaaattctt ccttcatctc ttccaagtca ag -            #ctgctgaa    600                                                                 - - aatcaaactt ccacgatgga gaaagatgtt cgtccaaaga caagaagtat aa -            #acagattt    660                                                                 - - cctcttcatt ctgaaagttc atcaaagaaa gcaccaagaa gcaaaaacaa aa -            #ccttggat    720                                                                 - - ccacttttga tgtcagagcc caagcccagg accagctccc ctagaggaga ta -            #aagcagcc    780                                                                 - - tatgacattc tgaggagatg ttctcagtgt ggcatcctgc ttcccctgcc ga -            #tcctaaat    840                                                                 - - caacatcagg agaaatgccg gtggttagct tcatcaaaaa ggaaaacaag tg -            #agaaattt    900                                                                 - - cagctagatt tggaaaagga aaggtactac aaattcaaaa gatttcactt tt -            #aacactgg    960                                                                 - - cattcctgcc tacttgctgt ggtggtcttg tgaaaggtga tgggttttat tc -            #gttgggct   1020                                                                 - - ttaaaagaaa aggtttggca gaactaaaaa caaaactcac gtatcatctc aa -            #tagataca   1080                                                                 - - gaaaaggctt ttgataaaat tcaacttgac ttcatgttaa aaaccctcaa ca -            #aaccaggc   1140                                                                 - - gtcgaaggaa catacctcaa aataataaga gccatctatg acaaaaccac ag -            #ccaacatc   1200                                                                 - - atactgaatg agcaaaagct ggagcattac tcttgagaag tagaacaagg ca -            #cttcagtc   1260                                                                 - - ctattcaaca tagtactgga agtctcgcca cagcaatcag gcaagagaaa ga -            #agtaaaag   1320                                                                 - - gcaccc                 - #                  - #                  -     #         1326                                                                  - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 317                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 2                                                         - - Met Glu Gly Asp Phe Ser Val Cys Arg Asn Cy - #s Lys Arg His Val Val       1               5  - #                10  - #                15               - - Ser Ala Asn Phe Thr Leu His Glu Ala Tyr Cy - #s Leu Arg Phe Leu Val                  20      - #            25      - #            30                   - - Leu Cys Pro Glu Cys Glu Glu Pro Val Pro Ly - #s Glu Thr Met Glu Glu              35          - #        40          - #        45                       - - His Cys Lys Leu Glu His Gln Gln Val Gly Cy - #s Thr Met Cys Gln Gln          50              - #    55              - #    60                           - - Ser Met Gln Lys Ser Ser Leu Glu Phe His Ly - #s Ala Asn Glu Cys Gln      65                  - #70                  - #75                  - #80        - - Glu Arg Pro Val Glu Cys Lys Phe Cys Lys Le - #u Asp Met Gln Leu Ser                      85  - #                90  - #                95               - - Lys Leu Glu Leu His Glu Ser Tyr Cys Gly Se - #r Arg Thr Glu Leu Cys                  100      - #           105      - #           110                  - - Gln Gly Cys Gly Gln Phe Ile Met His Arg Me - #t Leu Ala Gln His Arg              115          - #       120          - #       125                      - - Asp Val Cys Arg Ser Glu Gln Ala Gln Leu Gl - #y Lys Gly Glu Arg Ile          130              - #   135              - #   140                          - - Ser Ala Pro Glu Arg Glu Ile Tyr Cys His Ty - #r Cys Asn Gln Met Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Glu Asn Lys Tyr Phe His His Met Gly Ly - #s Cys Cys Pro Asp        Ser                                                                                             165  - #               170  - #               175             - - Glu Phe Lys Lys His Phe Pro Val Gly Asn Pr - #o Glu Ile Leu Pro Ser                  180      - #           185      - #           190                  - - Ser Leu Pro Ser Gln Ala Ala Glu Asn Gln Th - #r Ser Thr Met Glu Lys              195          - #       200          - #       205                      - - Asp Val Arg Pro Lys Thr Arg Ser Ile Asn Ar - #g Phe Pro Leu His Ser          210              - #   215              - #   220                          - - Glu Ser Ser Ser Lys Lys Ala Pro Arg Ser Ly - #s Asn Lys Thr Leu Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Pro Leu Leu Met Ser Glu Pro Lys Pro Arg Th - #r Ser Ser Pro Arg        Gly                                                                                             245  - #               250  - #               255             - - Asp Lys Ala Ala Tyr Asp Ile Leu Arg Arg Cy - #s Ser Gln Cys Gly Ile                  260      - #           265      - #           270                  - - Leu Leu Pro Leu Pro Ile Leu Asn Gln His Gl - #n Glu Lys Cys Arg Trp              275          - #       280          - #       285                      - - Leu Ala Ser Ser Lys Arg Lys Thr Ser Glu Ly - #s Phe Gln Leu Asp Leu          290              - #   295              - #   300                          - - Glu Lys Glu Arg Tyr Tyr Lys Phe Lys Arg Ph - #e His Phe                  305                 3 - #10                 3 - #15                            - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 1311                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 3                                                         - - gcagctagtg tgtcatttca gcgtttctcc tctcgtccct ggaagagcta aa -             #gatggctg     60                                                                 - - aatttctaga tgaccaggaa actcgactgt gtgacaactg caaaaaagaa at -            #tcctgtgt    120                                                                 - - ttaactttac catccatgag atccactgtc aaaggaacat tggtatgtgt cc -            #tacctgta    180                                                                 - - aggaaccatt tcccaaatct gacatggaga ctcacatggc tgcagaacac tg -            #tcaggtga    240                                                                 - - cctgcaaatg taacaagaag ttggagaaga ggctgttaaa gaagcatgag ga -            #gactgagt    300                                                                 - - gccctttgcg gcttgctgtc tgccagcact gtgatttaga actttccatt ct -            #caaactga    360                                                                 - - aggaacatga agattattgt ggtgcccgga cggaactatg tggcaactgt gg -            #tcgcaatg    420                                                                 - - tccttgtgaa agatctgaag actcaccctg aagtttgtgg gagagagggg ga -            #ggaaaaga    480                                                                 - - gaaatgaggt tgccatacct cctaatgcat atgatgaatc ttggggtcag ga -            #tggaatct    540                                                                 - - ggattgcatc ccaactcctc agacaaattg aggctctgga cccacccatg ag -            #gctgccgc    600                                                                 - - gaaggcccct gagagccttt gaatcagatg ttttccacaa tagaactacc aa -            #ccaaagga    660                                                                 - - acattacagc ccaggtttca attcagaata atctgtttga agaacaagag ag -            #gcaggaaa    720                                                                 - - ggaatagagg ccaacagccc cccaaagagg gtggtgaaga gagtgcaaac tt -            #ggacttca    780                                                                 - - tgttggccct aagtctgcaa aatgaaggcc aagcctccag tgtggcagag ca -            #ggacttct    840                                                                 - - ggagggccgt atgtgaggcc gaccagtctc atggcggtcc caggtctctc ag -            #tgacataa    900                                                                 - - agggtgcagc tgacgagatc atgttgcctt gtgaattttg tgaggagctc ta -            #cccagagg    960                                                                 - - aactgctgat tgaccatcag acaagctgta acccttcacg tgccttacct tc -            #actcaata   1020                                                                 - - ctggcagctc ttcccccaga ggggtggagg aacctgatgt catcttccag aa -            #ctccttgc   1080                                                                 - - aacaggctgc aagtaaccag ttagactctt tgatgggcct gagcaattca ca -            #ccctgtgg   1140                                                                 - - aggagagcat cattatccca tgtgaattct gtggggtaca gctggaagag ga -            #ggtgctgt   1200                                                                 - - tccatcacca ggaccagtgt gaccaacgcc cagccactgc aaccaaccat gt -            #gacagagg   1260                                                                 - - ggattcctag actggattcc cagcctcaag agccccttcc ccttgttttt a - #               1311                                                                        - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 419                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 4                                                         - - Met Ala Glu Phe Leu Asp Asp Gln Glu Thr Ar - #g Leu Cys Asp Asn Cys       1               5  - #                10  - #                15               - - Lys Lys Glu Ile Pro Val Phe Asn Phe Thr Il - #e His Glu Ile His Cys                  20      - #            25      - #            30                   - - Gln Arg Asn Ile Gly Met Cys Pro Thr Cys Ly - #s Glu Pro Phe Pro Lys              35          - #        40          - #        45                       - - Ser Asp Met Glu Thr His Met Ala Ala Glu Hi - #s Cys Gln Val Thr Cys          50              - #    55              - #    60                           - - Lys Cys Asn Lys Lys Leu Glu Lys Arg Leu Le - #u Lys Lys His Glu Glu      65                  - #70                  - #75                  - #80        - - Thr Glu Cys Pro Leu Arg Leu Ala Val Cys Gl - #n His Cys Asp Leu Glu                      85  - #                90  - #                95               - - Leu Ser Ile Leu Lys Leu Lys Glu His Glu As - #p Tyr Cys Gly Ala Arg                  100      - #           105      - #           110                  - - Thr Glu Leu Cys Gly Asn Cys Gly Arg Asn Va - #l Leu Val Lys Asp Leu              115          - #       120          - #       125                      - - Lys Thr His Pro Glu Val Cys Gly Arg Glu Gl - #y Glu Glu Lys Arg Asn          130              - #   135              - #   140                          - - Glu Val Ala Ile Pro Pro Asn Ala Tyr Asp Gl - #u Ser Trp Gly Gln Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Ile Trp Ile Ala Ser Gln Leu Leu Arg Gl - #n Ile Glu Ala Leu        Asp                                                                                             165  - #               170  - #               175             - - Pro Pro Met Arg Leu Pro Arg Arg Pro Leu Ar - #g Ala Phe Glu Ser Asp                  180      - #           185      - #           190                  - - Val Phe His Asn Arg Thr Thr Asn Gln Arg As - #n Ile Thr Ala Gln Val              195          - #       200          - #       205                      - - Ser Ile Gln Asn Asn Leu Phe Glu Glu Gln Gl - #u Arg Gln Glu Arg Asn          210              - #   215              - #   220                          - - Arg Gly Gln Gln Pro Pro Lys Glu Gly Gly Gl - #u Glu Ser Ala Asn Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Phe Met Leu Ala Leu Ser Leu Gln Asn Gl - #u Gly Gln Ala Ser        Ser                                                                                             245  - #               250  - #               255             - - Val Ala Glu Gln Asp Phe Trp Arg Ala Val Cy - #s Glu Ala Asp Gln Ser                  260      - #           265      - #           270                  - - His Gly Gly Pro Arg Ser Leu Ser Asp Ile Ly - #s Gly Ala Ala Asp Glu              275          - #       280          - #       285                      - - Ile Met Leu Pro Cys Glu Phe Cys Glu Glu Le - #u Tyr Pro Glu Glu Leu          290              - #   295              - #   300                          - - Leu Ile Asp His Gln Thr Ser Cys Asn Pro Se - #r Arg Ala Leu Pro Ser      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Leu Asn Thr Gly Ser Ser Ser Pro Arg Gly Va - #l Glu Glu Pro Asp        Val                                                                                             325  - #               330  - #               335             - - Ile Phe Gln Asn Ser Leu Gln Gln Ala Ala Se - #r Asn Gln Leu Asp Ser                  340      - #           345      - #           350                  - - Leu Met Gly Leu Ser Asn Ser His Pro Val Gl - #u Glu Ser Ile Ile Ile              355          - #       360          - #       365                      - - Pro Cys Glu Phe Cys Gly Val Gln Leu Glu Gl - #u Glu Val Leu Phe His          370              - #   375              - #   380                          - - His Gln Asp Gln Cys Asp Gln Arg Pro Ala Th - #r Ala Thr Asn His Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Thr Glu Gly Ile Pro Arg Leu Asp Ser Gln Pr - #o Gln Glu Pro Leu        Pro                                                                                             405  - #               410  - #               415             - - Leu Val Phe                                                               - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 1169                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 5                                                         - - tgggtgccag cccagctctc cttgtgtgcc gaagctcagc aactcagaca gc -             #caggacat     60                                                                 - - ccaggggcgg aatcgagaca gccagaatgg ggccatagcc cctgggcacg tt -            #tcagtgat    120                                                                 - - tcgccctcct caaaatctct acccagaaaa cattgtgccc tctttctccc gt -            #gggccttc    180                                                                 - - agggagatac ggagctagtg gtaggagtga aggtggcagg aattcccggg tc -            #acccctgc    240                                                                 - - agctgccaac taccgcagca gaactgcaaa ggcaaagcct tccaagcaac ag -            #ggagctgg    300                                                                 - - ggatgcagaa gaggaagagg aggagtaatg gtgtctccag agactttaca tc -            #ggttcctg    360                                                                 - - tcttctgtgc acagcagcac ttgccgctgt gcaggcccac ctctttggct ct -            #ttgggtgg    420                                                                 - - gagagttttt ccagatttta gatttttcta ggttatggcc attttgtgtc tt -            #ttgaggtt    480                                                                 - - gtgctgtggg ggtttgggtt tgagggaagg gagcagggtg gcggttgagg aa -            #cgcttcag    540                                                                 - - ccttagctgc tacctttcgg cagcagtgaa atacaagctg cagcctcggc tg -            #ccagggct    600                                                                 - - cccttttgac ttattgtcgc cactgcccct tggtgctgtg tggtcccagt gg -            #aaggaggg    660                                                                 - - gaagattttg gaaacctggt agccaccagt aaggtgattc tctgccctgt tg -            #gggcctaa    720                                                                 - - atttgggggc ttttgggcaa cctctccgtg tactgcgtct gtccacactc ga -            #ttgggccc    780                                                                 - - caggtgtgta tgaggcgctc tggtaaggtg ctcaggccag ttgcaatgtc tg -            #tcagtaac    840                                                                 - - gaggcttttg atgtgttgag ctggaggtga gtggaccggg ggctgtgttt ta -            #agctgctt    900                                                                 - - ccttggcatt tggcatcact gccttctgtt cccgggggag catggatctt tt -            #gtcctcac    960                                                                 - - tgctttctaa tggggagggc tgagggctcc ctgtccccac agcaggtatg gt -            #tgctctgc   1020                                                                 - - cccagcccca cacttgctct gaaaaccaag tgtcagagcc ccttcccctt gt -            #ttttattt   1080                                                                 - - tactgttata ataattatta acttccttgt aatagaaata aagtttgtac tt -            #ggaaaaaa   1140                                                                 - - aaaaaaaaaa aaaaaaaaaa aaaaaaaaa         - #                  - #              1169                                                                     - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 178                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 6                                                         - - Met Glu Gly Asp Phe Ser Val Cys Arg Asn Cy - #s Lys Arg His Val Val       1               5  - #                10  - #                15               - - Ser Ala Asn Phe Thr Leu His Glu Ala Tyr Cy - #s Leu Arg Phe Leu Val                  20      - #            25      - #            30                   - - Leu Cys Pro Glu Cys Glu Glu Pro Val Pro Ly - #s Glu Thr Met Glu Glu              35          - #        40          - #        45                       - - His Cys Lys Leu Glu His Gln Gln Val Gly Cy - #s Thr Met Cys Gln Gln          50              - #    55              - #    60                           - - Ser Met Gln Lys Ser Ser Leu Glu Phe His Ly - #s Ala Asn Glu Cys Gln      65                  - #70                  - #75                  - #80        - - Glu Arg Pro Val Glu Cys Lys Phe Cys Lys Le - #u Asp Met Gln Leu Ser                      85  - #                90  - #                95               - - Lys Leu Glu Leu His Glu Ser Tyr Cys Gly Se - #r Arg Thr Glu Leu Cys                  100      - #           105      - #           110                  - - Gln Gly Cys Gly Gln Phe Ile Met His Arg Me - #t Leu Ala Gln His Arg              115          - #       120          - #       125                      - - Asp Val Cys Arg Ser Glu Gln Ala Gln Leu Gl - #y Lys Gly Glu Arg Ile          130              - #   135              - #   140                          - - Ser Ala Pro Glu Arg Glu Ile Tyr Cys His Ty - #r Cys Asn Gln Met Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Glu Asn Lys Tyr Phe His His Met Gly Ly - #s Cys Cys Pro Asp        Ser                                                                                             165  - #               170  - #               175             - - Glu Phe                                                                   - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 172                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 7                                                         - - Met Glu Gly Asp Phe Ser Val Cys Arg Asn Cy - #s Lys Arg His Val Val       1               5  - #                10  - #                15               - - Ser Ala Asn Phe Thr Leu His Glu Ala Tyr Cy - #s Leu Arg Phe Leu Val                  20      - #            25      - #            30                   - - Leu Cys Pro Glu Cys Glu Glu Pro Val Pro Ly - #s Glu Thr Met Glu Glu              35          - #        40          - #        45                       - - His Cys Lys Leu Glu His Gln Gln Val Gly Cy - #s Thr Met Cys Gln Gln          50              - #    55              - #    60                           - - Ser Met Gln Lys Ser Ser Leu Glu Phe His Ly - #s Ala Asn Glu Cys Gln      65                  - #70                  - #75                  - #80        - - Glu Arg Pro Val Glu Cys Lys Phe Cys Lys Le - #u Asp Met Gln Leu Ser                      85  - #                90  - #                95               - - Lys Leu Glu Leu His Glu Ser Tyr Cys Gly Se - #r Arg Thr Glu Leu Cys                  100      - #           105      - #           110                  - - Gln Gly Cys Gly Gln Phe Ile Met His Arg Me - #t Leu Ala Gln His Arg              115          - #       120          - #       125                      - - Asp Val Cys Arg Ser Glu Gln Ala Gln Leu Gl - #y Lys Gly Glu Arg Ile          130              - #   135              - #   140                          - - Ser Ala Pro Glu Arg Glu Ile Tyr Cys His Ty - #r Cys Asn Gln Met Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Glu Asn Lys Tyr Phe His His Met Gly Ly - #s Cys                                      165  - #               170                                     - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 145                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 8                                                         - - Cys Pro Asp Ser Glu Phe Lys Lys His Phe Pr - #o Val Gly Asn Pro        Glu                                                                              1               5  - #                10  - #                15              - - Ile Leu Pro Ser Ser Leu Pro Ser Gln Ala Al - #a Glu Asn Gln Thr Ser                  20      - #            25      - #            30                   - - Thr Met Glu Lys Asp Val Arg Pro Lys Thr Ar - #g Ser Ile Asn Arg Phe              35          - #        40          - #        45                       - - Pro Leu His Ser Glu Ser Ser Ser Lys Lys Al - #a Pro Arg Ser Lys Asn          50              - #    55              - #    60                           - - Lys Thr Leu Asp Pro Leu Leu Met Ser Glu Pr - #o Lys Pro Arg Thr Ser      65                  - #70                  - #75                  - #80        - - Ser Pro Arg Gly Asp Lys Ala Ala Tyr Asp Il - #e Leu Arg Arg Cys Ser                      85  - #                90  - #                95               - - Gln Cys Gly Ile Leu Leu Pro Leu Pro Ile Le - #u Asn Gln His Gln Glu                  100      - #           105      - #           110                  - - Lys Cys Arg Trp Leu Ala Ser Ser Lys Arg Ly - #s Thr Ser Glu Lys Phe              115          - #       120          - #       125                      - - Gln Leu Asp Leu Glu Lys Glu Arg Tyr Tyr Ly - #s Phe Lys Arg Phe His          130              - #   135              - #   140                          - - Phe                                                                      145                                                                            - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 2643                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 9                                                         - - gcagctagtg tgtcatttca gcgtttctcc tctcgtccct ggaagagcta aa -             #gatggctg     60                                                                 - - aatttctaga tgaccaggaa actcgactgt gtgacaactg caaaaaagaa at -            #tcctgtgt    120                                                                 - - ttaactttac catccatgag atccactgtc aaaggaacat tggtatgtgt cc -            #tacctgta    180                                                                 - - aggaaccatt tcccaaatct gacatggaga ctcacatggc tgcagaacac tg -            #tcaggtga    240                                                                 - - cctgcaaatg taacaagaag ttggagaaga ggctgttaaa gaagcatgag ga -            #gactgagt    300                                                                 - - gccctttgcg gcttgctgtc tgccagcact gtgatttaga actttccatt ct -            #caaactga    360                                                                 - - aggaacatga agattattgt ggtgcccgga cggaactatg tggcaactgt gg -            #tcgcaatg    420                                                                 - - tccttgtgaa agatctgaag actcaccctg aagtttgtgg gagagagggg ga -            #ggaaaaga    480                                                                 - - gaaatgaggt tgccatacct cctaatgcat atgatgaatc ttggggtcag ga -            #tggaatct    540                                                                 - - ggattgcatc ccaactcctc agacaaattg aggctctgga cccacccatg ag -            #gctgccgc    600                                                                 - - gaaggcccct gagagccttt gaatcagatg ttttccacaa tagaactacc aa -            #ccaaagga    660                                                                 - - acattacagc ccaggtttca attcagaata atctgtttga agaacaagag ag -            #gcaggaaa    720                                                                 - - ggaatagagg ccaacagccc cccaaagagg gtggtgaaga gagtgcaaac tt -            #ggacttca    780                                                                 - - tgttggccct aagtctgcaa aatgaaggcc aagcctccag tgtggcagag ca -            #ggacttct    840                                                                 - - ggagggccgt atgtgaggcc gaccagtctc atggcggtcc caggtctctc ag -            #tgacataa    900                                                                 - - agggtgcagc tgacgagatc atgttgcctt gtgaattttg tgaggagctc ta -            #cccagagg    960                                                                 - - aactgctgat tgaccatcag acaagctgta acccttcacg tgccttacct tc -            #actcaata   1020                                                                 - - ctggcagctc ttcccccaga ggggtggagg aacctgatgt catcttccag aa -            #cttcttgc   1080                                                                 - - aacaggctgc aagtaaccag ttagactctt tgatgggcct gagcaattca ca -            #ccctgtgg   1140                                                                 - - aggagagcat cattatccca tgtgaattct gtggggtaca gctggaagag ga -            #ggtgctgt   1200                                                                 - - tccatcacca ggaccagtgt gaccaacgcc cagccactgc aaccaaccat gt -            #gacagagg   1260                                                                 - - ggattcctag actggattcc cagcctcaag agaccccacc agagctgccc ag -            #gaggcgtg   1320                                                                 - - tcagacacca gggagacctg tcttctggtt acctggatga tactaagcag ga -            #aacagcta   1380                                                                 - - atgggcccac ctcctgtctg cctcccagcc gacccattaa caatatgaca gc -            #tacctata   1440                                                                 - - accagctatc gagatcaaca tcaggcccca gacctgggtg ccagcccagc tc -            #tccttgtg   1500                                                                 - - tgccgaagct cagcaactca gacagccagg acatccaggg gcggaatcga ga -            #cagccaga   1560                                                                 - - atggggccat agcccctggg cacgtttcag tgattcgccc tcctcaaaat ct -            #ctacccag   1620                                                                 - - aaaacattgt gccctctttc tcccctgggc cttcagggag atacggagct ag -            #tggtagga   1680                                                                 - - gtgaaggtgg caggaattcc cgggtcaccc ctgcagctgc caactaccgc ag -            #cagaactg   1740                                                                 - - caaaggcaaa gccttccaag caacagggag ctggggatgc agaagaggaa ga -            #ggaggagt   1800                                                                 - - aatggtgtct ccagagactt tacatcggtt cctgtcttct gtgcacagca gc -            #acttgccg   1860                                                                 - - ctgtgcaggc ccacctcttt ggctctttgg gtgggagagt ttttccagat tt -            #tagatttt   1920                                                                 - - tctaggttat ggccattttg tgtcttttga ggttgtgctg tgggggtttg gg -            #tttgaggg   1980                                                                 - - aagggagcag ggtggcggtt gaggaacgct tcagccttag ctgctacctt tc -            #ggcagcag   2040                                                                 - - tgaaatacaa gctgcagcct cggctgccag ggctcccttt tgacttattg tc -            #gccactgc   2100                                                                 - - cccttggtgc tgtgtggtcc cagtggaagg aggggaagat tttggaaacc tg -            #gtagccac   2160                                                                 - - cagtaaggtg attctctgcc ctgttggggc ctaaatttgg gggcttttgg gc -            #aacctctc   2220                                                                 - - cgtgtactgc gtctgtccac actcgattgg gccccaggtg tgtatgaggc gc -            #tctggtaa   2280                                                                 - - ggtgctcagg ccagttgcaa tgtctgtcag taacgaggct tttgatgtgt tg -            #agctggag   2340                                                                 - - gtgagtggac cgggggctgt gttttaagct gcttccttgg catttggcat ca -            #ctgccttc   2400                                                                 - - tgttcccggg ggagcatgga tcttttgtcc tcactgcttt ctaatgggga gg -            #gctgaggg   2460                                                                 - - ctccctgtcc ccacagcagg tatggttgct ctgccccagc cccacacttg ct -            #ctgaaaac   2520                                                                 - - caagtgtcag agccccttcc ccttgttttt attttactgt tataataatt at -            #taacttcc   2580                                                                 - - ttgtaataga aataaagttt gtacttggaa aaaaaaaaaa aaaaaaaaaa aa -            #aaaaaaaa   2640                                                                 - - aaa                  - #                  - #                  - #               2643                                                                  - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 582                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 10                                                        - - Met Ala Glu Phe Leu Asp Asp Gln Glu Thr Ar - #g Leu Cys Asp Asn Cys       1               5  - #                10  - #                15               - - Lys Lys Glu Ile Pro Val Phe Asn Phe Thr Il - #e His Glu Ile His Cys                  20      - #            25      - #            30                   - - Gln Arg Asn Ile Gly Met Cys Pro Thr Cys Ly - #s Glu Pro Phe Pro Lys              35          - #        40          - #        45                       - - Ser Asp Met Glu Thr His Met Ala Ala Glu Hi - #s Cys Gln Val Thr Cys          50              - #    55              - #    60                           - - Lys Cys Asn Lys Lys Leu Glu Lys Arg Leu Le - #u Lys Lys His Glu Glu      65                  - #70                  - #75                  - #80        - - Thr Glu Cys Pro Leu Arg Leu Ala Val Cys Gl - #n His Cys Asp Leu Glu                      85  - #                90  - #                95               - - Leu Ser Ile Leu Lys Leu Lys Glu His Glu As - #p Tyr Cys Gly Ala Arg                  100      - #           105      - #           110                  - - Thr Glu Leu Cys Gly Asn Cys Gly Arg Asn Va - #l Leu Val Lys Asp Leu              115          - #       120          - #       125                      - - Lys Thr His Pro Glu Val Cys Gly Arg Glu Gl - #y Glu Glu Lys Arg Asn          130              - #   135              - #   140                          - - Glu Val Ala Ile Pro Pro Asn Ala Tyr Asp Gl - #u Ser Trp Gly Gln Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Ile Trp Ile Ala Ser Gln Leu Leu Arg Gl - #n Ile Glu Ala Leu        Asp                                                                                             165  - #               170  - #               175             - - Pro Pro Met Arg Leu Pro Arg Arg Pro Leu Ar - #g Ala Phe Glu Ser Asp                  180      - #           185      - #           190                  - - Val Phe His Asn Arg Thr Thr Asn Gln Arg As - #n Ile Thr Ala Gln Val              195          - #       200          - #       205                      - - Ser Ile Gln Asn Asn Leu Phe Glu Glu Gln Gl - #u Arg Gln Glu Arg Asn          210              - #   215              - #   220                          - - Arg Gly Gln Gln Pro Pro Lys Glu Gly Gly Gl - #u Glu Ser Ala Asn Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Phe Met Leu Ala Leu Ser Leu Gln Asn Gl - #u Gly Gln Ala Ser        Ser                                                                                             245  - #               250  - #               255             - - Val Ala Glu Gln Asp Phe Trp Arg Ala Val Cy - #s Glu Ala Asp Gln Ser                  260      - #           265      - #           270                  - - His Gly Gly Pro Arg Ser Leu Ser Asp Ile Ly - #s Gly Ala Ala Asp Glu              275          - #       280          - #       285                      - - Ile Met Leu Pro Cys Glu Phe Cys Glu Glu Le - #u Tyr Pro Glu Glu Leu          290              - #   295              - #   300                          - - Leu Ile Asp His Gln Thr Ser Cys Asn Pro Se - #r Arg Ala Leu Pro Ser      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Leu Asn Thr Gly Ser Ser Ser Pro Arg Gly Va - #l Glu Glu Pro Asp        Val                                                                                             325  - #               330  - #               335             - - Ile Phe Gln Asn Phe Leu Gln Gln Ala Ala Se - #r Asn Gln Leu Asp Ser                  340      - #           345      - #           350                  - - Leu Met Gly Leu Ser Asn Ser His Pro Val Gl - #u Glu Ser Ile Ile Ile              355          - #       360          - #       365                      - - Pro Cys Glu Phe Cys Gly Val Gln Leu Glu Gl - #u Glu Val Leu Phe His          370              - #   375              - #   380                          - - His Gln Asp Gln Cys Asp Gln Arg Pro Ala Th - #r Ala Thr Asn His Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Thr Glu Gly Ile Pro Arg Leu Asp Ser Gln Pr - #o Gln Glu Thr Pro        Pro                                                                                             405  - #               410  - #               415             - - Glu Leu Pro Arg Arg Arg Val Arg His Gln Gl - #y Asp Leu Ser Ser Gly                  420      - #           425      - #           430                  - - Tyr Leu Asp Asp Thr Lys Gln Glu Thr Ala As - #n Gly Pro Thr Ser Cys              435          - #       440          - #       445                      - - Leu Pro Pro Ser Arg Pro Ile Asn Asn Met Th - #r Ala Thr Tyr Asn Gln          450              - #   455              - #   460                          - - Leu Ser Arg Ser Thr Ser Gly Pro Arg Pro Gl - #y Cys Gln Pro Ser Ser      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Pro Cys Val Pro Lys Leu Ser Asn Ser Asp Se - #r Gln Asp Ile Gln        Gly                                                                                             485  - #               490  - #               495             - - Arg Asn Arg Asp Ser Gln Asn Gly Ala Ile Al - #a Pro Gly His Val Ser                  500      - #           505      - #           510                  - - Val Ile Arg Pro Pro Gln Asn Leu Tyr Pro Gl - #u Asn Ile Val Pro Ser              515          - #       520          - #       525                      - - Phe Ser Pro Gly Pro Ser Gly Arg Tyr Gly Al - #a Ser Gly Arg Ser Glu          530              - #   535              - #   540                          - - Gly Gly Arg Asn Ser Arg Val Thr Pro Ala Al - #a Ala Asn Tyr Arg Ser      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Arg Thr Ala Lys Ala Lys Pro Ser Lys Gln Gl - #n Gly Ala Gly Asp        Ala                                                                                             565  - #               570  - #               575             - - Glu Glu Glu Glu Glu Glu                                                              580                                                                - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 1302                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 11                                                        - - gcagctagtg tgtcatttca gcgtttctcc tctcgtccct ggaagagcta aa -             #gatggctg     60                                                                 - - aatttctaga tgaccaggaa actcgactgt gtgacaactg caaaaaagaa at -            #tcctgtgt    120                                                                 - - ttaactttac catccatgag atccactgtc aaaggaacat tggtatgtgt cc -            #tacctgta    180                                                                 - - aggaaccatt tcccaaatct gacatggaga ctcacatggc tgcagaacac tg -            #tcaggtga    240                                                                 - - cctgcaaatg taacaagaag ttggagaaga ggctgttaaa gaagcatgag ga -            #gactgagt    300                                                                 - - gccctttgcg gcttgctgtc tgccagcact gtgatttaga actttccatt ct -            #caaactga    360                                                                 - - aggtcacccc tgcagctgcc aactaccgca gcagaactgc aaaggcaaag cc -            #ttccaagc    420                                                                 - - aacagggagc tggggatgca gaagaggaag aggaggagta atggtgtctc ca -            #gagacttt    480                                                                 - - acatcggttc ctgtcttctg tgcacagcag cacttgccgc tgtgcaggcc ca -            #cctctttg    540                                                                 - - gctctttggg tgggagagtt tttccagatt ttagattttt ctaggttatg gc -            #cattttgt    600                                                                 - - gtcttttgag gttgtgctgt gggggtttgg gtttgaggga agggagcagg gt -            #ggcggttg    660                                                                 - - aggaacgctt cagccttagc tgctaccttt cggcagcagt gaaatacaag ct -            #gcagcctc    720                                                                 - - ggctgccagg gctccctttt gacttattgt cgccactgcc ccttggtgct gt -            #gtggtccc    780                                                                 - - agtggaagga ggggaagatt ttggaaacct ggtagccacc agtaaggtga tt -            #ctctgccc    840                                                                 - - tgttggggcc taaatttggg ggcttttggg caacctctcc gtgtactgcg tc -            #tgtccaca    900                                                                 - - ctcgattggg ccccaggtgt gtatgaggcg ctctggtaag gtgctcaggc ca -            #gttgcaat    960                                                                 - - gtctgtcagt aacgaggctt ttgatgtgtt gagctggagg tgagtggacc gg -            #gggctgtg   1020                                                                 - - ttttaagctg cttccttggc atttggcatc actgccttct gttcccgggg ga -            #gcatggat   1080                                                                 - - cttttgtcct cactgctttc taatggggag ggctgagggc tccctgtccc ca -            #cagcaggt   1140                                                                 - - atggttgctc tgccccagcc ccacacttgc tctgaaaacc aagtgtcaga gc -            #cccttccc   1200                                                                 - - cttgttttta ttttactgtt ataataatta ttaacttcct tgtaatagaa at -            #aaagtttg   1260                                                                 - - tacttggaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa    - #                      - #1302                                                                     - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 135                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 12                                                        - - Met Ala Glu Phe Leu Asp Asp Gln Glu Thr Ar - #g Leu Cys Asp Asn Cys       1               5  - #                10  - #                15               - - Lys Lys Glu Ile Pro Val Phe Asn Phe Thr Il - #e His Glu Ile His Cys                  20      - #            25      - #            30                   - - Gln Arg Asn Ile Gly Met Cys Pro Thr Cys Ly - #s Glu Pro Phe Pro Lys              35          - #        40          - #        45                       - - Ser Asp Met Glu Thr His Met Ala Ala Glu Hi - #s Cys Gln Val Thr Cys          50              - #    55              - #    60                           - - Lys Cys Asn Lys Lys Leu Glu Lys Arg Leu Le - #u Lys Lys His Glu Glu      65                  - #70                  - #75                  - #80        - - Thr Glu Cys Pro Leu Arg Leu Ala Val Cys Gl - #n His Cys Asp Leu Glu                      85  - #                90  - #                95               - - Leu Ser Ile Leu Lys Leu Lys Val Thr Pro Al - #a Ala Ala Asn Tyr Arg                  100      - #           105      - #           110                  - - Ser Arg Thr Ala Lys Ala Lys Pro Ser Lys Gl - #n Gln Gly Ala Gly Asp              115          - #       120          - #       125                      - - Ala Glu Glu Glu Glu Glu Glu                                                  130              - #   135                                                 - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 19                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(19)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d; Xaa at 16 and 19 is       Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 13                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa                                                               - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(20)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 17 and 20 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 14                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa                                                                      20                                                                 - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 21                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(21)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 18 and 21 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 15                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa                                                                  20                                                                 - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 19 and 22 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 16                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(20)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 17 and 20 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 17                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa                                                                      20                                                                 - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 21                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(21)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 19 and 21 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 18                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa                                                                  20                                                                 - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 19 and 22 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 19                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 20 and 23 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 20                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 21 and 24 is      Cys                                                                                  or His                                                                    - - <400> SEQUENCE: 21                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 22 and 25 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 22                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 23 and 26 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 23                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 21                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(21)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 21 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 24                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa                                                                  20                                                                 - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 25                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 26                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 27                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 27                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 28                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 28                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 29                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 29                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 30                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 30                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 31                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 31                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 32                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 32                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 33                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d  Xaa at 21 and 24 is       Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 33                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 34                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 34                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 35                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 35                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 36                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 36                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 37                                                  <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 37                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 38                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 38                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 39                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 39                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 40                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 40                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 41                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 41                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 42                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 42                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 43                                                  <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 43                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 44                                                  <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 26 and 29 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 44                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 45                                                  <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(20)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 16 and 20 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 45                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa                                                                      20                                                                 - -  - - <210> SEQ ID NO 46                                                  <211> LENGTH: 21                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(21)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 17 and 21 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 46                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa                                                                  20                                                                 - -  - - <210> SEQ ID NO 47                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 17 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 47                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 48                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 48                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 49                                                  <211> LENGTH: 21                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(21)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 17 and 21 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 49                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa                                                                  20                                                                 - -  - - <210> SEQ ID NO 50                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 18 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 50                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 51                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 51                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 52                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 52                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 53                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 53                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 54                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 54                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 55                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 55                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 56                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 18 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 56                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 57                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 57                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 58                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 58                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 59                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 59                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 60                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 60                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 61                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 61                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 62                                                  <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 62                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 63                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 63                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 64                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 64                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 65                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 65                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 66                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 66                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 67                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 67                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 68                                                  <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 68                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 69                                                  <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 29 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 69                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 70                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 70                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 71                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 71                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 72                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 72                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 73                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 73                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 74                                                  <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 74                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 75                                                  <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 29 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 75                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 76                                                  <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(30)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 26 and 30 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 76                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa                          20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 77                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(21)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 16 and 21 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 77                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 78                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 17 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 78                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 79                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 18 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 79                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 80                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 80                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 81                                                  <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 17 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 81                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 82                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 18 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 82                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 83                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 83                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 84                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 84                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 85                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 85                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 86                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 22 and 27 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 86                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 87                                                  <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 87                                                        - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 88                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 16 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 88                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 89                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 89                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 90                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 20 and 25 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 90                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 91                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 91                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 92                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 92                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 93                                                  <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 93                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 94                                                  <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 24 and 29 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 94                                                        - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 95                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 95                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 96                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 96                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 97                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 97                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 98                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 98                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 99                                                  <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 99                                                        - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 100                                                 <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 29 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 100                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 101                                                 <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(30)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 30 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 101                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa                          20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 102                                                 <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 102                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 103                                                 <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 21 and 26 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 103                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 104                                                 <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 104                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 105                                                 <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 23 and 28 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 105                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 106                                                 <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;  Xaa at 24 and 29 is      Cys                                                                                   or His                                                                   - - <400> SEQUENCE: 106                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                              1               5  - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 107                                                 <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(30)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 30 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 107                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa                          20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 108                                                 <211> LENGTH: 31                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(31)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 26 and 31 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 108                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa                      20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 109                                                 <211> LENGTH: 22                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(22)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 17 and 22 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 109                                                       - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa                                                              20                                                                 - -  - - <210> SEQ ID NO 110                                                 <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 18 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 110                                                       - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 111                                                 <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 111                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 112                                                 <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 112                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 113                                                 <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(23)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 18 and 23 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 113                                                       - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                          20                                                                 - -  - - <210> SEQ ID NO 114                                                 <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 114                                                       - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 115                                                 <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 115                                                       - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 116                                                 <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 116                                                       - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 117                                                 <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 117                                                       - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 118                                                 <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 118                                                       - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 119                                                 <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 29 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 119                                                       - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 120                                                 <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(24)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 19 and 24 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 120                                                       - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                      20                                                                 - -  - - <210> SEQ ID NO 121                                                 <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 121                                                       - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 122                                                 <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 122                                                       - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 123                                                 <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 123                                                       - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 124                                                 <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 124                                                       - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 125                                                 <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 29 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 125                                                       - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 126                                                 <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(30)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 30 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 126                                                       - - Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa                          20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 127                                                 <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(25)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 20 and 25 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 127                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                  20      - #            25                                          - -  - - <210> SEQ ID NO 128                                                 <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 128                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 129                                                 <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 129                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 130                                                 <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 23 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 130                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 131                                                 <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 24 and 29 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 131                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 132                                                 <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(30)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 30 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 132                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa                          20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 133                                                 <211> LENGTH: 31                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(31)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 26 and 31 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 133                                                       - - Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa                      20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 134                                                 <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(26)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 21 and 26 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 134                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                              20      - #            25                                          - -  - - <210> SEQ ID NO 135                                                 <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(27)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 27 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 135                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                      20      - #            25                                          - -  - - <210> SEQ ID NO 136                                                 <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(28)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 22 and 28 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 136                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa                                  20      - #            25                                          - -  - - <210> SEQ ID NO 137                                                 <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(29)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 29 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 137                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa                              20      - #            25                                          - -  - - <210> SEQ ID NO 138                                                 <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(30)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 25 and 30 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 138                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa                          20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 139                                                 <211> LENGTH: 31                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(31)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 26 and 31 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 139                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa                      20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 140                                                 <211> LENGTH: 32                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(32)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d;   Xaa at 27 and 32 is     Cys                                                                                  or His                                                                   - - <400> SEQUENCE: 140                                                       - - Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa                  20      - #            25      - #            30                   - -  - - <210> SEQ ID NO 141                                                 <211> LENGTH: 19                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(19)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d                              - - <400> SEQUENCE: 141                                                       - - Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa His Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Cys                                                               - -  - - <210> SEQ ID NO 142                                                 <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(20)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d                              - - <400> SEQUENCE: 142                                                       - - Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa His Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Cys                                                                      20                                                                 - -  - - <210> SEQ ID NO 143                                                 <211> LENGTH: 21                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)...(21)                                                    <223> OTHER INFORMATION: Xaa = Any Amino Aci - #d                              - - <400> SEQUENCE: 143                                                       - - Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa His Xaa       1               5  - #                10  - #                15               - - Xaa Xaa Xaa Xaa Cys                                                                  20                                                               __________________________________________________________________________

What is claimed is:
 1. A substantially pure nucleic acid encoding amammalian XAF polypeptide, wherein said polypeptide has at least 50%sequence identity to SEQ ID NO: 2, 4, 10, or
 12. 2. The nucleic acid ofclaim 1 wherein said nucleic acid is genomic DNA or cDNA.
 3. The nucleicacid of claim 1 wherein said nucleic acid is operably linked toregulatory sequences for expression of said polypeptide and wherein saidregulatory sequences comprise a promoter.
 4. The nucleic acid of claim 1wherein said mammalian XAF polypeptide is selected from a groupconsisting of the XAF-1 amino terminal 131 amino acids, and the XAF-2amino terminal 135 amino acids.
 5. The nucleic acid of claim 1, whereinsaid polypeptide has at least 50% sequence identity to SEQ ID NO:
 2. 6.The nucleic acid of claim 1, wherein said polypeptide has at least 50%sequence identity to SEQ ID NO:
 10. 7. The nucleic acid of claim 1,wherein said polypeptide has at least 50% sequence identity to SEQ IDNO:
 12. 8. The nucleic acid of claim 1, wherein said polypeptide has atleast 50% sequence identity to SEQ ID NO:
 4. 9. A substantially purenucleic acid comprising a nucleic acid sequence corresponding to atleast ten nucleotides of a nucleic acid encoding a mammalian XAFpolypeptide, wherein said nucleic acid is antisense nucleic acidsufficient to decrease mammalian XAF biological activity.
 10. Theantisense nucleic acid of claim 9, said antisense nucleic acidcorresponding to at least fifteen nucleotides of a nucleic acid encodinga XAF polypeptide.
 11. The antisense nucleic acid of claim 9, whereinsaid XAF biological activity is decreased by at least 20%.
 12. Theantisense nucleic acid of claim 9, wherein said antisense nucleic acidis encoded by a vector directing expression of said antisense nucleicacid in a vector-containing cell.
 13. The nucleic acid of claim 4,wherein said amino-terminal 135 amino acids has the amino acid sequenceof SEQ ID NO.: 4 or the nucleic acid sequence of SEQ ID NO.:
 3. 14. Thenucleic acid of claim 5, wherein said XAF-1 has the amino acid sequenceof SEQ ID NO.: 2 or the nucleic acid sequence of SEQ ID NO.:
 1. 15. Thenucleic acid of claim 5, wherein said XAF-1 has a deletion of thenucleic acids encoding the carboxy terminal amino acids 173 to
 317. 16.The nucleic acid of claim 5, wherein said XAF-1 has a deletion of thenucleic acids encoding the amino terminal amino acids 1 to
 172. 17. Thenucleic acid of claim 6, wherein said XAF-2L has the amino acid sequenceof SEQ ID NO.: 10 or the nucleic acid sequence of SEQ ID NO.:
 9. 18. Thenucleic acid of claim 7, wherein said XAF-2S has the amino acid sequenceof SEQ ID NO.:
 12. 19. A vector comprising a substantially pure nucleicacid encoding a mammalian XAF polypeptide, said vector directingexpression of said polypeptide in a vector-containing cell, and saidpolypeptide having at least 50% sequence identity to SEQ ID NO: 2, 4,10, or
 12. 20. A cell that contains a substantially pure nucleic acidencoding a mammalian XAF polypeptide, wherein said polypeptide has atleast 50% sequence identity to SEQ ID NO: 2, 4, 10, or
 12. 21. Thenucleic acid of claim 1, 5, 6, 7, or 9, wherein said nucleic acid ishuman.
 22. The nucleic acid of claim 1, 5, 6, 7, 9, 19, or 20, wheresaid nucleic acid encodes a polypeptide having at least one of thefollowing biological activities: the ability to bind an IAP; or theability to bind another XAF polypeptide; or the ability to causeapoptosis when transfected into a cell; or the ability to enhance theNF-κB inducing activity of a TRAF.
 23. The nucleic acid of claim 1, 5,6, 7, 9, 19, or 20, wherein said nucleic acid encodes a polypeptidewhich binds to a XAF, XAF-1, XAF-2L, or XAF-2S specific antibody.
 24. Asubstantially pure nucleic acid which hybridizes under high stringencyconditions to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 1, 3, 9, or
 11. 25. The nucleic acid of claim 24, whereinsaid nucleic acid hybridizes under high stringency conditions to thenucleic acid sequence of SEQ ID NO:
 1. 26. The nucleic acid of claim 24,wherein said nucleic acid hybridizes under high stringency conditions tothe nucleic acid sequence of SEQ ID NO:
 3. 27. The nucleic acid of claim24, wherein said nucleic acid hybridizes under high stringencyconditions to the nucleic acid sequence of SEQ ID NO:
 9. 28. The nucleicacid of claim 24, wherein said nucleic acid hybridizes under highstringency conditions to the nucleic acid sequence of SEQ ID NO: 11.